Viral complement control proteins for eye disorders

ABSTRACT

The present invention provides compositions and methods for treating and/or preventing age related macular degeneration and other conditions involving macular degeneration or choroidal neovascularization. Certain of the compositions comprise a poxvirus complement control protein or a complement binding fragment or variant thereof. Other compositions comprise a poxvirus complement control protein linked to a moiety that binds to a component present on or at the surface of cell or noncellular molecular entity, e.g., a component present in the eye of a subject at risk of or suffering from age related macular degeneration or a related condition or choroidal neovascularization. Certain of the methods comprise administering a poxvirus complement control protein or complement binding fragment or variant thereof to a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to provisional application 60/616,983,filed Oct. 8, 2004, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The macula is a small area in the retina of the eye, approximately 3 to5 millimeters in size, adjacent to the optic nerve. It is the mostsensitive area of the retina and contains the fovea, a depressed regionthat allows for high visual acuity and contains a dense concentration ofcones, the photoreceptors that are responsible for color vision.

Macular degeneration is a term that refers to a number of differentdiseases characterized by degenerative changes in the macula, all ofwhich lead to a loss of central vision. Age-related macular degeneration(ARMD) is the most common cause of functional blindness in developedcountries for those over 50 years of age (Seddon, J M. Epidemiology ofage-related macular degeneration. In: Ogden, T E, et al., eds. Ryan S J,ed-in-chief. Retina Vol II. 3rd ed. St. Louis, Mo.: Mosby;2001:1039-50). The disease is characterized by progressive degenerationof the retina, retinal pigment epithelium (RPE), and underlying choroid(the highly vascular tissue that lies beneath the RPE, between theretina and the sclera). The retinal pigment epithelial layer is believedto be crucial for photoreceptor health. Cells in this layer recyclevisual pigment (rhodopsin), phagocytose photoreceptor tips daily as partof rod and cone regeneration, and transport fluid across the membrane tothe choroid, which is believed to help prevent detachment of the neuralretina. Central vision deteriorates when cells in the RPE cease tofunction properly, which can lead to photoreceptor degeneration.

Despite extensive investigation, the pathogenesis of ARMD remainsunclear, and the etiology of the molecular events that occur is not wellunderstood. A variety of factors including oxidative stress,inflammation with a possible autoimmune component, genetic background(e.g., mutations), and environmental or behavioral features such assmoking and diet may contribute to the pathogenesis of ARMD in ways thatare as yet poorly understood. Regardless of the underlying etiology, aclinical hallmark of ARMD is the appearance of drusen, localizeddeposits of lipoproteinaceous material that accumulate in the spacebetween the RPE and Bruch's membrane, which separates the RPE from thechoroidal vessels (choriocapillaris). Drusen are typically the earliestclinical finding in ARMD, and the existence, location, and number ofdrusen are used in classifying the disease into stages and formonitoring its progression (Ambati, J., et al., Surv. Ophthalmol.,48(3): 257-293, 2003; “Preferred Practice Pattern: Age-Related MacularDegeneration”, American Academy of Ophthalmology, 2003). Drusen aretypically the earliest clinical finding in ARMD.

ARMD has been classified into both “dry” and “wet” (exudative, orneovascular) forms. Dry ARMD is much more common than wet ARMD, but thedry form can progress to the wet form, and the two occur simultaneouslyin a significant number of cases. Dry ARMD is typically characterized byprogressive apoptosis of cells in the RPE layer, overlying photoreceptorcells, and frequently also the underlying cells in the choroidalcapillary layer. Confluent areas (typically at least 175 μm in minimumdiameter) of RPE cell death accompanied by overlying photoreceptoratrophy are referred to as geographic atrophy. Patients with this formof ARMD experience a slow and progressive deterioration in centralvision.

Wet ARMD is characterized by bleeding and/or leakage of fluid fromabnormal vessels that have grown from the choroidal vessels(choriocapillaris) beneath the RPE and the macula, which can beresponsible for sudden and disabling loss of vision. It has beenestimated that much of the vision loss that patients experience is dueto such choroidal neovascularization (CNV) and its secondarycomplications. A subtype of neovascular ARMD in which angiomatousproliferation originates from the retina and extends posteriorly intothe subretinal space, eventually communicating in some cases withchoroidal new vessels has been identified (Yannuzzi, L. A., et al.,Retina, 21(5):416-34, 2001). This form of neovascular ARMD, termedretinal angiomatous proliferation (RAP) can be particularly severe. Theexistence of macular drusen is a strong risk factor for the developmentof both wet and dry forms of ARMD (Ambati, J., et al., supra).

Treatment options for ARMD are limited, and none are fully effective(Ambati, J., et al., Surv. Ophthalmol., 48(3): 257-293, 2003, andreferences therein). Thus there is a need in the art for new approachesto the treatment of ARMD and also of other diseases and conditions ofthe eye characterized by macular degeneration, choroidalneovascularization, retinal neovascularization, retinal angiomatousproliferation, and/or blood vessel leakage. Such diseases and conditionsinclude, but are not limited to, diabetic retinopathy and retinopathy ofprematurity.

SUMMARY OF THE INVENTION

The present invention addresses the foregoing needs, among others. Theinvention provides a method of treating an eye disorder characterized bymacular degeneration, choroidal neovascularization, retinalneovascularization, or any combination of these, comprising (i)providing a subject in need of treatment for the eye disorder; and (ii)administering a composition comprising a viral complement controlprotein (VCCP) or a complement inhibiting fragment or variant thereof tothe subject.

The invention further provides a method of treating an eye disordercharacterized by macular degeneration, choroidal neovascularization,retinal neovascularization, or any combination of these, comprising (i)providing a subject in need of treatment for the eye disorder; and (ii)administering a composition comprising a viral complement interferingprotein (VCIP) or a complement inhibiting fragment or variant thereof tothe subject.

The invention further provides a composition comprising: (i) a VCCP or acomplement inhibiting fragment or variant thereof; and (ii) a moietythat binds to a component present in the eye of a subject at risk of orsuffering from an eye disorder characterized by macular degeneration,choroidal neovascularization, retinal neovascularization, or anycombination of these. The invention further provides a compositioncomprising: (i) a VCIP or a complement inhibiting fragment or variantthereof; and (ii) a moiety that binds to a component present in the eyeof a subject at risk of or suffering from an eye disorder characterizedby macular degeneration, choroidal neovascularization, retinalneovascularization, or any combination of these. In the foregoingcompositions, the component can be a cellular marker or a noncellularentity, e.g., a molecule or complex that is present in deposits found inthe eye of a subject with macular degeneration.

The invention further provides a composition comprising: (i) a VCCP or acomplement inhibiting fragment or variant thereof; and (ii) anangiogenesis inhibitor. The invention further provides a compositioncomprising: (i) a VCIP or a complement inhibiting fragment or variantthereof; and (ii) an angiogenesis inhibitor.

The invention further provides a composition comprising: (i) a VCCP or acomplement inhibiting fragment or variant thereof; and (ii) a solublegel-forming material. The composition forms a gel following introductioninto the body, e.g., upon contact with a physiological fluid. Theinvention further provides a composition comprising: (i) a VCIP or acomplement inhibiting fragment or variant thereof; and (ii) a solublegel-forming material. The composition forms a gel following introductioninto the body, e.g., upon contact with a physiological fluid. In certainembodiments of the invention any of the compositions comprising asoluble gel-forming material further comprises an angiogenesisinhibitor. The composition may be formed into a gel implant in vitro andadministered to or in the vicinity of the eye.

The invention further provides ocular and periocular implants andpolymeric delivery vehicles comprising (i) a VCCP or a complementinhibiting fragment or variant thereof or (ii) a VCIP or a complementinhibiting fragment or variant thereof. In some embodiments of theinvention the composition further comprises a moiety that binds to acomponent present in the eye of a subject at risk of or suffering froman eye disorder characterized by macular degeneration, choroidalneovascularization, retinal neovascularization, or any combination ofthese. In certain embodiments of the invention either of the foregoingcompositions further comprises an angiogenesis inhibitor.

The invention further provides multimeric complexes comprising two ormore different VCCPs or VCIPs. The invention further provides asupramolecular complex comprising at least one VCCP or VCIP.

The invention further provides methods of treating an eye disordercharacterized by macular degeneration, choroidal neovascularization,retinal neovascularization, or any combination of these, comprisingadministering any of the inventive compositions to a subject at risk ofor suffering from the eye disorder. The compositions can be administeredas sole therapy or one or more other treatments for the disorder mayalso be administered either concurrently or sequentially. Suchtreatments include, but are not limited to, laser photocoagulation,photodynamic therapy (e.g., Visudyne®), or anti-angiogenic therapy.

Methods for testing the inventive compositions and methods are alsoprovided.

Methods for making the inventive compositions are also provided.

In any of the embodiments of the present invention, the eye disorder canbe a macular degeneration related condition, diabetic retinopathy,retinopathy of prematurity, or any condition featuring choroidal and/orretinal neovascularization.

Included among the eye disorders that can be treated with thecompositions and methods of the invention are eye disorders in whichretinal angiomatous proliferation (RAP) is present. RAP involvesabnormal proliferation of retinal blood vessels (retinalneovascularization) and is a feature of a subtype of neovascular ARMD,but the compositions and methods of the invention can be used to treatRAP due to any cause, whether or not associated with maculardegeneration. The invention therefore provides a method of inhibiting aneye disorder characterized by retinal angiomatous proliferationcomprising (i) providing a subject in need of treatment for the eyedisorder; and (ii) administering a composition comprising a VCCP, aVCIP, or a complement inhibiting fragment or variant of either, to thesubject. The composition can be administered using any of the methodsdescribed herein. In some embodiments the composition is delivered inclose proximity to the posterior segment of the eye.

In any of the embodiments of the invention the VCCP can be a poxvirusVCCP (PVCCP) or a herpesvirus VCCP (HVCCP). In any of the embodiments ofthe invention, the PVCCP can be from vaccinia virus, variola virus,monkeypox virus, cowpox virus, etc. In any of the embodiments of theinvention involving a fragment or variant of a VCCP, the fragment orvariant may be at least 80% identical to the VCCP, at least 85%identical to the VCCP, at least 90% identical to the VCCP, or at least95% identical to the VCCP, provided that the fragment or variantinhibits complement. In any of the embodiments of the inventioninvolving a fragment or variant of a VCIP, the fragment or variant maybe at least at least 80% identical to the VCIP, at least 85% identicalto the VCIP, at least 90% identical to the VCIP, or at least 95%identical to the VCIP, provided that the fragment or variant inhibitscomplement.

In any of the embodiments of the invention that features an angiogenesisinhibitor, the angiogenesis inhibitor may be any angiogenesis inhibitorknown in the art. For example, the angiogenesis inhibitor may, but neednot be, selected from the group consisting of: Macugen® or another VEGFnucleic acid ligand; Lucentis®, Avastin®, or another anti-VEGF antibody;combretastatin or a derivative or prodrug thereof such as CombretastatinA4 Prodrug (CA4P); VEGF-Trap; EVIZON™ (squalamine lactate); AG-013958(Pfizer, Inc.); JSM6427 (Jerini AG), β2-glycoprotein 1 (β2-GP1), and ashort interfering RNA (siRNA) that inhibits expression of one or moreVEGF isoforms (e.g., VEGF₁₆₅) or inhibits expression of a VEGF receptor(e.g., VEGFR1).

Unless otherwise stated, the invention makes use of standard methods ofmolecular biology, cell culture, animal maintenance, ophthalmologicexamination, and administration of therapeutic agents to subjects, etc.,and uses art-accepted meanings of terms. This application refers tovarious patents and publications. The contents of all scientificarticles, books, patents, and other publications, mentioned in thisapplication are incorporated herein by reference. In addition, thefollowing publications are incorporated herein by reference: CurrentProtocols in Molecular Biology, Current Protocols in Immunology, CurrentProtocols in Protein Science, and Current Protocols in Cell Biology, allJohn Wiley & Sons, N.Y., edition as of July 2002; Sambrook, Russell, andSambrook, Molecular Cloning: A Laboratory Manual, 3^(rd) ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 2001; KubyImmunology, 4^(th) ed., Goldsby, R. A., Kindt, T. J., and Osborne, B.(eds.), W.H. Freeman, 2000, Goodman and Gilman's The PharmacologicalBasis of Therapeutics, 10^(th) Ed. McGraw Hill, 2001, Katzung, B. (ed.)Basic and Clinical Pharmacology, McGraw-Hill/Appleton & Lange; 9thedition (December 2003), Ophthalmic Surgery: Principles and Practice,3^(rd) ed., W.B. Saunders Company, 2002; Albert, D M and Lucarelli, M J(eds.), Clinical Atlas of Procedures in Ophthalmic Surgery, AmericanMedical Association, 2003. In the event of a conflict or inconsistencybetween any of the incorporated references and the instantspecification, the specification shall control, it being understood thatthe determination of whether a conflict or inconsistency exists iswithin the discretion of the inventors and can be made at any time.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-1E show schematic representations of the anterior and posteriorsegments of the eye (1A and 1B) and the outer layers of the eye (1C-1E).FIG. 1C depicts a normal eye. FIG. 1D depicts an eye suffering from dryARMD. FIG. 1E depicts an eye suffering from exudative ARMD. ONL=outernuclear layer; IS=inner segment; OS=outer segment; RPE=retinal pigmentepithelial layer; BM=Bruch's membrane; CC=choriocapillaris. From Tezel,T., et al., Trends in Molecular Medicine, 10(9), 417-420, 2004.

FIG. 2 shows a consensus sequence for a short consensus repeat (SCR), amodule found in complement control proteins. From Smith, S A, et al., J.Virol. 74(12), 5659-5666, 2000.

FIGS. 3A and 3B show sequences of vaccinia virus complement controlprotein precursor (SEQ ID NO: 1) and the mature vaccinia viruscomplement control protein (SEQ ID NO: 2).

FIG. 4 shows a sequence comparison of mature complement control proteinsfrom a variety of orthopoxvirus isolates (SEQ ID NO: 3-10). Thecorresponding genetic loci are as follows: SEQ ID NO: 3 is VAC-COP C3L,SEQ ID NO: 4 is VAC-WR C21L, SEQ ID NO: 5 is CPV-GRI C17L, SEQ ID NO: 6is CPV-BRI IMP, SEQ ID NO: 7 is VAR-BSH D15L, SEQ ID NO: 8 is VAR-INDD12L, SEQ ID NO: 9 is VAR-GAR B18L, SEQ ID NO: 10 is MPV-ZAI D15L.Modified from Smith, S A, et al., J. Virol. 74(12), 5659-5666, 2000.

FIG. 5 shows a comparison of the SCR domain structure of a number ofcomplement control proteins and fragments thereof, the number of K+Rresidues, %K+R residues, pI, number of putative heparin binding sites,and ability to inhibit hemolysis and/or bind to heparin. Modified fromSmith, S A, et al., J. Virol. 74(12), 5659-5666, 2000. The domains areSCR modules. Thus, for example, rVCP SCR (2, 3, 4), is a recombinantlyproduced polypeptide containing SCRs 2, 3, and 4 from VCP.

FIG. 6 shows the amino acid sequence of SPICE (SEQ ID NO: 12).

FIG. 7 shows confocal micrographs of CNV (stained green) in the eyes ofmice without (A) and with (B) injection of VCP.

FIG. 8 is a graph showing a comparison of the mean CNV area (in μm²) inmice that received either no treatment or received an intravitrealinjection of albumin, or an intravitreal injection of VCP (either 10 μgor 30 μg).

DEFINITIONS

“Angiogenesis” or “angiogenic” refer to formation, growth, and/ordevelopment of new blood vessels.

The terms “angiogenesis inhibitor” and “antiangiogenic agent” are usedinterchangeably herein to refer to agents that are capable of inhibitingor reducing one or more processes associated with angiogenesisincluding, but not limited to, endothelial cell proliferation,endothelial cell migration, and capillary tube formation.

The terms “approximately” or “about” in reference to a number aregenerally include numbers that fall within a range of 5% in eitherdirection (greater than or less than) of the number unless otherwisestated or otherwise evident from the context (except where such numberwould exceed 100% of a possible value).

“Biocompatible” refers to a material that is substantially non-toxic tocells in vitro, e.g., if its addition to cells in culture results inless than or equal to 20% cell death. A material is consideredbiocompatible with respect to a recipient if it is substantiallynontoxic to the recipient's cells in the quantities and at the locationused, and also does not elicit or cause a significant deleterious oruntoward effect on the recipient's body, e.g., an immunological orinflammatory reaction, unacceptable scar tissue formation, etc.

“Biodegradable” means that a material is capable of being broken downphysically and/or chemically within cells or within the body of asubject, e.g., by hydrolysis under physiological conditions, by naturalbiological processes such as the action of enzymes present within cellsor within the body, etc., to form smaller chemical species which can bemetabolized and, optionally, reused, and/or excreted or otherwisedisposed of. Preferably a biodegradable compound is biocompatible.

A “biological macromolecule” is a large molecule composed of smallersubunits of a type that are found in biological systems. Examples ofbiological macromolecules include polypeptides, nucleic acids, andpolysaccharides. Typically a biological macromolecule contains at least3 subunits (e.g., amino acids, nucleosides, monosaccharides, etc.). Thebiological macromolecule may, but need not be, a naturally occurringpolypeptide, nucleic acid, or polysaccharide. The biologicalmacromolecule may be modified, e.g., it may be conjugated to anonbiological molecule such as synthetic polymer, etc.

“Choroidal neovascularization” (CNV) refers to the abnormal development,proliferation, and/or growth of blood vessels arising from thechoriocapillaris. The blood vessels typically extend through Bruch'smembrane, RPE layer, and/or subretinal space.

A “complement component” or “complement protein” is a molecule that isinvolved in activation of the complement system or participates in oneor more complement-mediated activities. Components of the classicalcomplement pathway include, e.g., C1q, C1r, C1s, C2, C3, C4, C5, C6, C7,C8, C9, and the C5b-9 complex, also referred to as the membrane attackcomplex (MAC) and active fragments or enzymatic cleavage products of anyof the foregoing (e.g., C3a, C3b, C4a, C4b, C5a, etc.). Components ofthe alternative pathway include, e.g., factors B, D, H, and I, andproperdin.

A “complement-inhibiting fragment” of a VCCP or VCIP is a polypeptidefragment of the VCCP or VCIP that inhibits complement, e.g., thepolypeptide fragment inhibits complement activation.

A “complement-inhibiting variant” of a VCCP or VCIP is a polypeptidevariant of the VCCP or VCIP that inhibits complement, e.g., the variantinterferes with complement activation.

“Concurrent administration” as used herein with respect to two or moreagents, e.g., therapeutic agents, is administration performed usingdoses and time intervals such that the administered agents are presenttogether within the body, or at a site of action in the body such aswithin the eye) over a time interval in less than de minimis quantities.The time interval can be minutes (e.g., at least 1 minute, 1-30 minutes,30-60 minutes), hours (e.g., at least 1 hour, 1-2 hours, 2-6 hours, 6-12hours, 12-24 hours), days (e.g., at least 1 day, 1-2 days, 2-4 days, 4-7days, etc.), weeks (e.g., at least 1, 2, or 3 weeks, etc. Accordingly,the agents may, but need not be, administered together as part of asingle composition. In addition, the agents may, but need not be,administered simultaneously (e.g., within less than 5 minutes, or withinless than 1 minute) or within a short time of one another (e.g., lessthan 1 hour, less than 30 minutes, less than 10 minutes, approximately 5minutes apart). According to various embodiments of the invention agentsadministered within such time intervals may be considered to beadministered at substantially the same time. In certain embodiments ofthe invention concurrently administered agents are present at effectiveconcentrations within the body (e.g., in the blood and/or at a site ofaction such as the retina) over the time interval. When administeredconcurrently, the effective concentration of each of the agents neededto elicit a particular biological response may be less than theeffective concentration of each agent when administered alone, therebyallowing a reduction in the dose of one or more of the agents relativeto the dose that would be needed if the agent was administered as asingle agent. The effects of multiple agents may, but need not be,additive or synergistic. The agents may be administered multiple times.The de minimis concentration of an agent may be, for example, less thanapproximately 5% of the concentration that would be required to elicit aparticular biological response, e.g., a desired biological response.

An “effective amount” of an active agent refers to the amount of theactive agent sufficient to elicit a desired biological response. As willbe appreciated by those of ordinary skill in this art, the absoluteamount of a particular agent that is effective may vary depending onsuch factors as the desired biological endpoint, the agent to bedelivered, the target tissue, etc. Those of ordinary skill in the artwill further understand that an “effective amount” may be administeredin a single dose, or may be achieved by administration of multipledoses. For example, an effective amount may be an amount sufficient toachieve one or more of the following: (i) inhibit or prevent drusenformation; (ii) cause a reduction in drusen number and/or size (drusenregression); (iii) cause a reduction in or prevent lipofuscin deposits;(iv) inhibit or prevent visual loss or slow the rate of visual loss; (v)inhibit choroidal neovascularization or slow the rate of choroidalneovascularization; (vi) cause a reduction in size and/or number oflesions characterized by choroidal neovascularization; (vii) inhibitchoroidal neovascularization or slow the rate of retinalneovascularization; (viii) cause a reduction in size and/or number oflesions characterized by retinal neovascularization; (ix) improve visualacuity and/or contrast sensitivity; (x) inhibit or prevent photoreceptoror RPE cell atrophy or apoptosis, or reduce the rate of photoreceptor orRPE cell atrophy or apoptosis; (xi) inhibit or prevent progression ofnon-exudative macular degeneration to exudative macular degeneration.

An “expression control sequence” refers to a nucleotide sequence in apolynucleotide that regulates the expression (transcription and/ortranslation) of a nucleotide sequence operatively linked thereto.

“Exudative” macular degeneration is used herein synonymously with “wet”type macular degeneration, as those terms are generally understood inthe art, i.e., to refer to a macular degeneration related condition suchas ARMD characterized by neovascularization.

“Fibrillar collagen solids” means the dry collagen solid content offibrillar collagen. Fibrillar collagen is an insoluble collagen materialwherein the collagen molecules interact to form microfibrils whichthemselves aggregate by side-to-side and end-to-end association to formstabilized collagen fibrils.

“Fusion protein” refers to a polypeptide that contains two or moredifferent polypeptides or portions thereof joined together to form asingle polypeptide chain. A recombinant polynucleotide that encodes afusion protein may be created by removing the stop codon from thepolynucleotide that encodes the first polypeptide and appending apolynucleotide that encodes the second polypeptide in frame, so that theresulting recombinant polynucleotide encodes a single polypeptidecomprising the two polypeptides.

“Herpesvirus” refers to members of a family of complex, double-strandedDNA viruses constituting the family Herpesviridae. The family includesthe subfamilies Alphaherpesvirinae, which includes herpes simplexvirus-1 and -2 (HSV-1 and HSV-2), varicella zoster virus (VZV), bovineherpesvirus-1 (BHV-1), pseudorabies virus (PRV), and equineherpesvirus-1 and -3 (EHV-1 and EHV-4) and Gammaherpesvirinae, whichincludes Epstein Barr virus (EBV) and lymphotropic herpesvirus saimiri(HVS). The herpevirus family is described in Fields, B N, et al., FieldsVirology, 3rd ed., Lippincott Williams & Wilkins, 2001.

“Identity” refers to the extent to which the sequence of two or morenucleic acids or polypeptides is the same. The percent identity betweena sequence of interest and a second sequence over a window ofevaluation, e.g., over the length of the sequence of interest, may becomputed by aligning the sequences, determining the number of residues(nucleotides or amino acids) within the window of evaluation that areopposite an identical residue allowing the introduction of gaps tomaximize identity, dividing by the total number of residues of thesequence of interest or the second sequence (whichever is greater) thatfall within the window, and multiplying by 100. By gap is meant aportion of a sequence that is not occupied by a residue. For example,the sequence A K L - - - S I G (SEQ ID NO: 11) contains a gap of threeresidues. When computing the number of identical residues needed toachieve a particular percent identity, fractions are to be rounded tothe nearest whole number. Percent identity can be calculated with theuse of a variety of computer programs known in the art. For example,computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc.,generate alignments and provide percent identity between a sequence ofinterest and sequences in any of a variety of public databases. Thealgorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad.Sci. USA 87:22264-2268, 1990) modified as in Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5877, 1993 is incorporated into the NBLASTand XBLAST programs of Altschul et al. (Altschul, et al., J. Mol. Biol.215:403-410, 1990). To obtain gapped alignments for comparison purposes,Gapped BLAST is utilized as described in Altschul et al. (Altschul, etal. Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programsare used. A PAM250 or BLOSUM62 matrix may be used. See the Web sitehaving URL www. followed immediately by ncbi.nlm.nih.gov for theseprograms. In a specific embodiment, percent identity of a sequence ofinterest and a second sequence is calculated using BLAST2 with defaultparameters.

The term “isolated” means 1) separated from at least some of thecomponents with which it is usually associated in nature; 2) prepared orpurified by a process that involves the hand of man; and/or 3) notoccurring in nature. For example, a molecule that is removed from a cellthat produces it, is “isolated”. A chemically synthesized molecule is“isolated”.

The term “linked”, when used with respect to two or more moieties, meansthat the moieties are physically associated or connected with oneanother to form a molecular structure that is sufficiently stable sothat the moieties remain associated under the conditions in which thelinkage is formed and, preferably, under the conditions in which the newmolecular structure is used, e.g., physiological conditions. In certainpreferred embodiments of the invention the linkage is a covalentlinkage. In other embodiments the linkage is noncovalent. Moieties maybe linked either directly or indirectly. When two moieties are directlylinked, they are either covalently bonded to one another or are insufficiently close proximity such that intermolecular forces between thetwo moieties maintain their association. When two moieties areindirectly linked, they are each linked either covalently ornoncovalently to a third moiety, which maintains the association betweenthe two moieties. In general, when two moieties are referred to as beinglinked by a “linker” or “linking moiety” or “linking portion”, thelinkage between the two linked moieties is indirect, and typically eachof the linked moieties is covalently bonded to the linker. The linkercan be any suitable moiety that reacts with the two moieties to belinked within a reasonable period of time, under conditions consistentwith stability of the moieties (which may be protected as appropriate,depending upon the conditions), and in sufficient amount, to produce areasonable yield.

“Liposomes” are artificial microscopic spherical particles formed by alipid bilayer (or multilayers) enclosing an aqueous compartment.Liposomes are commonly used as a delivery vehicle for various types ofmolecules (such as proteins, small molecules, DNA, and RNA), including anumber of different drugs and can be used for delivering certain of thecompositions of the invention.

“Local administration” or “local delivery”, in reference to delivery ofa composition or agent of the invention, refers to delivery that doesnot rely upon transport of the composition or agent to its intendedtarget tissue or site via the vascular system. The composition or agentmay be delivered directly to its intended target tissue or site, or inthe vicinity thereof, e.g., in close proximity to the intended targettissue or site. For example, the composition may be delivered byinjection or implantation of the composition or agent or by injection orimplantation of a device containing the composition or agent. Followinglocal administration in the vicinity of a target tissue or site, thecomposition or agent, or one or more components thereof, may diffuse tothe intended target tissue or site. It will be understood that oncehaving been locally delivered a fraction of a therapeutic agent(typically only a minor fraction of the administered dose) may enter thevascular system and be transported to another location, including backto its intended target tissue or site.

“Macular degeneration related condition” refers to any of a number ofdisorders and conditions in which the macula degenerates or losesfunctional activity. The degeneration or loss of functional activity canarise as a result of, for example, cell death, decreased cellproliferation, loss of normal biological function, or a combination ofthe foregoing. Macular degeneration can lead to and/or manifest asalterations in the structural integrity of the cells and/orextracellular matrix of the macula, alteration in normal cellular and/orextracellular matrix architecture, and/or the loss of function ofmacular cells. The cells can be any cell type normally present in ornear the macula including RPE cells, photoreceptors, and/or capillaryendothelial cells. ARMD is the major macular degeneration relatedcondition, but a number of others are known including, but not limitedto, Best macular dystrophy, Sorsby fundus dystrophy, MallatiaLeventinese and Doyne honeycomb retinal dystrophy.

“Marker”, for the purpose of the description of the invention, may referto any molecular moiety (e.g., protein, peptide, mRNA or other RNAspecies, DNA, lipid, carbohydrate) that characterizes, indicates, oridentifies a particular diseased or physiological state (e.g.,apoptotic, cancerous, normal) or characterizes, indicates, or identifiesone or more cell type(s), tissue type(s), or embryological origin. Thepresence or absence of certain marker(s), or the amount of certainmarker(s), may indicate a particular physiological or diseased state ofa patient, organ, tissue, or cell. A cellular marker is a marker foundin or on a cell. A cellular marker may, but need not be, cell typespecific. For example, a cell type specific marker is generally aprotein, peptide, mRNA, lipid, or carbohydrate that is present at ahigher level on or in a particular cell type or cell types of interestthan on or in many other cell types. In some instances a cell typespecific marker is present at detectable levels only on or in aparticular cell type of interest. However, it will be appreciated thatuseful markers need not be absolutely specific for the cell type ofinterest. For example, certain CD molecules are present on the cells ofmultiple different types of leukocytes. In general, a cell type specificmarker for a particular cell type is expressed at levels at least 3 foldgreater in that cell type than in a reference population of cells whichmay consist, for example, of a mixture containing cells from a plurality(e.g., 5-10 or more) of different tissues or organs in approximatelyequal amounts. More preferably the cell type specific marker is presentat levels at least 4-5 fold, between 5-10 fold, or more than 10-foldgreater than its average expression in a reference population.Preferably detection or measurement of a cell type specific marker makesit possible to distinguish the cell type or types of interest from cellsof many, most, or all other types. In general, the presence and/orabundance of most markers may be determined using standard techniquessuch as Northern blotting, in situ hybridization, RT-PCR, sequencing,immunological methods such as immunoblotting, immunodetection, orfluorescence detection following staining with fluorescently labeledantibodies, oligonucleotide or cDNA microarray or membrane array,protein microarray analysis, mass spectrometry, etc.

“Non-exudative” macular degeneration is used herein synonymously with“dry” type macular degeneration as those terms are generally used in theart, to refer to a macular degeneration related condition, e.g., ARMD,in which neovascularization that would be detectable using standardmethods such as fluorescein angiography has not occurred.

“Operably linked” or “operably associated” refers to a relationshipbetween two nucleic acid sequences wherein the expression of one of thenucleic acid sequences is controlled by, regulated by, modulated by,etc., the other nucleic acid sequences, or a relationship between twopolypeptides wherein the expression of one of the polypeptides iscontrolled by, regulated by, modulated by, etc., the other polypeptide.For example, the transcription of a nucleic acid sequence is directed byan operably linked promoter sequence; post-transcriptional processing ofa nucleic acid is directed by an operably linked processing sequence;the translation of a nucleic acid sequence is directed by an operablylinked translational regulatory sequence; the transport, stability, orlocalization of a nucleic acid or polypeptide is directed by an operablylinked transport or localization sequence; and the post-translationalprocessing of a polypeptide is directed by an operably linked processingsequence. Preferably a nucleic acid sequence that is operably linked toa second nucleic acid sequence, or a polypeptide that is operativelylinked to a second polypeptide, is covalently linked, either directly orindirectly, to such a sequence, although any effective three-dimensionalassociation is acceptable.

“Plurality” means more than one.

“Polynucleotide” or “oligonucleotide” refers to a polymer ofnucleotides. As used herein, an oligonucleotide is typically less than100 nucleotides in length. A polynucleotide or oligonucleotide may alsobe referred to as a nucleic acid. Typically, a polynucleotide comprisesat least three nucleotides. A nucleotide comprises a nitrogenous base, asugar molecule, and a phosphate group. A nucleoside comprises anitrogenous base linked to a sugar molecule. In a polynucleotide oroligonucleotide, phosphate groups covalently link adjacent nucleosidesto form a polymer. The polymer may comprise or natural nucleosides foundin DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine),other nucleosides or nucleoside analogs, nucleosides containingchemically modified bases and/or biologically modified bases (e.g.,methylated bases), intercalated bases, modified sugars, etc. Thephosphate groups in a polynucleotide or oligonucleotide are typicallyconsidered to form the internucleoside backbone of the polymer. Innaturally occurring nucleic acids (DNA or RNA), the backbone linkage isvia a 3′ to 5′ phosphodiester bond. However, polynucleotides andoligonucleotides containing modified backbones or non-naturallyoccurring internucleoside linkages can also be used in the presentinvention. Such modified backbones include ones that have a phosphorusatom in the backbone and others that do not have a phosphorus atom inthe backbone. Examples of modified linkages include, but are not limitedto, phosphorothioate and 5′-N-phosphoramidite linkages. See Kornberg andBaker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992), Scheit,Nucleotide Analogs (John Wiley, New York, 1980), U.S. Patent Pub. No.20040092470 and references therein for further discussion of variousnucleotides, nucleosides, and backbone structures that can be used inthe polynucleotides or oligonucleotides described herein, and methodsfor producing them. Typically a polynucleotide of this invention is DNAor RNA.

Polynucleotides and oligonucleotides need not be uniformly modifiedalong the entire length of the molecule. For example, differentnucleotide modifications, different backbone structures, etc., may existat various positions in the polynucleotide or oligonucleotide. Any ofthe polynucleotides described herein may utilize these modifications.

The polynucleotide may be of any size or sequence and may be single- ordouble-stranded. If single-stranded the polynucleotide may be the coding(sense) strand or non-coding (anti-sense) strand.

The polynucleotide may be provided by any means known in the art. Incertain embodiments, the polynucleotide has been engineered usingrecombinant techniques (for a more detailed description of thesetechniques, please see Ausubel et al. Current Protocols in MolecularBiology (John Wiley & Sons, Inc., New York, 1999); Molecular Cloning: ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (ColdSpring Harbor Laboratory Press: 1989). The polynucleotide may also beobtained from natural sources and purified from contaminating componentsfound normally in nature. The polynucleotide may be synthesized usingenzymatic techniques, either within cells or in vitro. Thepolynucleotide may also be chemically synthesized in a laboratory, e.g.,using standard solid phase chemistry. The polynucleotide may be modifiedby chemical and/or biological means. In certain preferred embodiments,these modifications lead to increased stability of the polynucleotide.Modifications include methylation, phosphorylation, end-capping, etc.

The term “polynucleotide sequence” or “nucleic acid sequence” as usedherein can refer to the nucleic acid material itself and is notrestricted to the sequence information (i.e. the succession of letterschosen among the five base letters A, G, C, T, or U) that biochemicallycharacterizes a specific nucleic acid, e.g., a DNA or RNA molecule. Anucleic acid sequence is presented in the 5′ to 3′ direction unlessotherwise indicated.

“Polypeptide”, as used herein, refers to a polymer of amino acids. Aprotein is a molecule composed of one or more polypeptides. A peptide isa relatively short polypeptide, typically between about 2 and 60 aminoacids in length. The terms “protein”, “polypeptide”, and “peptide” maybe used interchangeably. Polypeptides used herein typically containamino acids such as those that are naturally found in proteins. However,amino acids that are not naturally found in proteins (i.e., amino acidsthat either do or do not occur in nature and that can be incorporatedinto a polypeptide chain), and/or amino acid analogs can also oralternatively be used. One or more of the amino acids in a polypeptidemay be modified, for example, by the addition of a chemical entity suchas a carbohydrate group, a phosphate group, a farnesyl group, anisofarnesyl group, a fatty acid group, a linker for conjugation,functionalization, or other modification, etc. In certain embodimentsthe modification(s) lead to a more stable polypeptide (e.g., greaterhalf-life in vivo or in vitro under conditions approximatingphysiological conditions) or a polypeptide having higher biologicalactivity. Modifications may include cyclization of the peptide, theincorporation of D-amino acids, etc. Preferably the modification doesnot substantially interfere with the desired biological activity of thepolypeptide. The natural or other chemical modifications such as thosedescribed above can occur anywhere in a polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. A given polypeptide may contain many types of modifications.Polypeptides may be branched or they may be cyclic, with or withoutbranching. Polypeptides may be conjugated with, encapsulated by, orembedded within a polymer or polymeric matrix, dendrimer, nanoparticle,microparticle, liposome, or the like.

Polypeptides of use in this invention (e.g., a VCCP, VCIP, collagen,etc.) may, for example, be purified from natural sources, produced invitro or in vivo in suitable expression systems using recombinant DNAtechnology in suitable expression systems (e.g., by recombinant hostcells or in transgenic animals or plants), synthesized through chemicalmeans such as conventional solid phase peptide synthesis and/or usingmethods involving chemical ligation of synthesized peptides (see, e.g.,Kent, S., J Pept Sci., 9(9):574-93, 2003 and U.S. Pub. No. 20040115774),or any combination of these.

The term “polypeptide sequence” or “amino acid sequence” as used hereincan refer to the polypeptide material itself and is not restricted tothe sequence information (i.e. the succession of letters or three lettercodes chosen among the letters and codes used as abbreviations for aminoacid names) that biochemically characterizes a polypeptide. Apolypeptide sequence presented herein is presented in an N-terminal toC-terminal direction unless otherwise indicated.

“Poxvirus” refers to a family of complex, double-stranded DNA virusesconstituting the family Poxviridae. The family includes theorthopoxviruses, a genus of the family Poxviridae, subfamilyChordopoxvirinae, comprising many species infecting mammals, includinghuman beings. Poxviruses are described in Fields, B N, et al., FieldsVirology, 3^(rd) ed., Lippincott Williams & Wilkins, 2001.Orthopoxviruses include, but are not limited to, vaccinia virus, variolavirus major, variola virus minor, cowpox virus, monkeypox virus,camelpox virus, swinepox virus, and ectromelia virus.

“Poxvirus complement control protein” refers to members of a family ofhomologous proteins encoded by a number of different poxviruses thatbind to one or more complement pathway proteins and inhibit either theclassical pathway of complement activation, the alternative pathway ofcomplement activation, the lectin pathway, or any combination of these.Poxvirus complement control proteins are members of the complementcontrol protein (CCP), also called regulators of complement activation(RCA) superfamily (Reid, K B M and Day, A J, Immunol Today, 10: 177-80,1989).

“Posterior segment of the eye” refers to the portion of the eye behindthe lens, including the vitreous, choroid, and retina (including themacula).

“Purified”, as used herein, means that an entity or substance isseparated from one or more other entities or substances with which itwas previously found before being purified. An entity or substance maybe partially purified, substantially purified, or pure. A substance orentity such as a nucleic acid or polypeptide is considered pure when itis removed from substantially all other compounds or entities other thana solvent and any ions contained in the solvent, i.e., it constitutes atleast about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or greater than 99% of the dry weight of thecomposition. A partially or substantially purified compound or entitysuch as a nucleic acid or polypeptide may be removed from at least 50%,at least 60%, at least 70%, or at least 80% by weight of the materialwith which it is naturally found, e.g., cellular material such ascellular proteins and/or nucleic acids. In certain embodiments the of apurified nucleic acid or polypeptide constitutes at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or even more, by dry weight, ofthe total nucleic acid or polypeptide, respectively, in a composition.Methods for assessing purity are known in the art and includechromatographic methods, immunological methods, electrophoretic methods,etc. Any of the polynucleotides or polypeptides described herein may bepurified.

“Recombinant host cells”, “host cells”, and other such terms, denoteprokaryotic or eukaryotic cells or cell lines that have been used asrecipients for an exogenous nucleic acid (typically DNA) such as anexpression vector into which a nucleic acid portion that encodes apolypeptide of interest has been inserted. These terms include theprogeny of the original cell into which the vector or other nucleic acidhas been introduced. Appropriate unicellular host cells include any ofthose routinely used in expressing polynucleotides (e.g., eukaryotic,mammalian, and/or viral polynucleotides) including, for example,prokaryotes, such as E. coli; and eukaryotes, including for example,fungi, such as yeast (e.g., Pichia pastoris); insect cells (e.g., Sf9),plant cells, and animal cells, e.g., mammalian cells such as CHO, R1.1,B-W, L-M, African Green Monkey Kidney cells (e.g. COS-1, COS-7, BSC-1,BSC-40 and BMT-10) and cultured human cells. Terms such as “host cells”,etc., are also used to refer to cells or cell lines that can be used asrecipients for an exogenous nucleic acid, prior to its introduction. A“recombinant polynucleotide” is one that contains nucleic acid portionsthat are not found joined together in nature. A “recombinantpolypeptide” is a polypeptide that is produced by transcription andtranslation of an exogenous nucleic acid by a recombinant host cell,typically after introduction of an expression vector that contains aportion that encodes the recombinant polypeptide into the host cell.

The term “regulatory element” or “regulatory sequence” in reference to anucleic acid is generally used herein to describe a portion of nucleicacid that regulates one or more steps in the expression (particularlytranscription, but in some cases other events such as splicing or otherprocessing) of nucleic acid sequence(s) with which it is operativelylinked. The term includes promoters and can also refer to enhancers andother transcriptional control elements. Promoters are regions of nucleicacid that include a site to which RNA polymerase binds before initiatingtranscription and that are typically necessary for even basal levels oftranscription to occur. Such elements frequently comprise a TATA box.Enhancers are regions of nucleic acid that encompass binding sites forprotein(s) that elevate transcriptional activity of a nearby ordistantly located promoter, typically above some basal level ofexpression that would exist in the absence of the enhancer. In someembodiments of the invention, regulatory sequences may directconstitutive expression of a nucleotide sequence (e.g., expression inmost or all cell types under typical physiological conditions in cultureor in an organism); in other embodiments, regulatory sequences maydirect cell or tissue-specific and/or inducible expression. For example,expression may be induced by the presence or addition of an inducingagent such as a hormone or other small molecule, by an increase intemperature, etc. Regulatory elements may also inhibit or decreaseexpression of an operatively linked nucleic acid. Such regulatoryelements may be referred to as “negative regulatory elements”.

“Retinal neovascularization” refers to the abnormal development,proliferation, and/or growth of blood vessels on or in the retina, e.g.,on the retinal surface.

“Sequential administration” of two or more agents refers toadministration of two or more agents to a subject such that the agentsare not present together in the subject's body at greater than deminimis concentrations. Administration of the agents may, but need not,alternate. Each agent may be administered multiple times.

“Small molecule” refers to organic compounds, whethernaturally-occurring or artificially created (e.g., via chemicalsynthesis) that have relatively low molecular weight and that are notproteins, polypeptides, or nucleic acids. Typically, small moleculeshave a molecular weight of less than about 1500 g/mol. Also, smallmolecules typically have multiple carbon-carbon bonds.

“Specific binding” generally refers to a physical association between atarget polypeptide (or, more generally, a target molecule) and a bindingmolecule such as an antibody or ligand. The association is typicallydependent upon the presence of a particular structural feature of thetarget such as an antigenic determinant or epitope recognized by thebinding molecule. For example, if an antibody is specific for epitope A,the presence of a polypeptide containing epitope A or the presence offree unlabeled A in a reaction containing both free labeled A and thebinding molecule that binds thereto, will reduce the amount of labeled Athat binds to the binding molecule. It is to be understood thatspecificity need not be absolute but generally refers to the context inwhich the binding occurs. For example, it is well known in the art thatnumerous antibodies cross-react with other epitopes in addition to thosepresent in the target molecule. Such cross-reactivity may be acceptabledepending upon the application for which the antibody is to be used. Oneof ordinary skill in the art will be able to select antibodies orligands having a sufficient degree of specificity to performappropriately in any given application (e.g., for detection of a targetmolecule, for therapeutic purposes, etc). It is also to be understoodthat specificity may be evaluated in the context of additional factorssuch as the affinity of the binding molecule for the target versus theaffinity of the binding molecule for other targets, e.g., competitors.If a binding molecule exhibits a high affinity for a target moleculethat it is desired to detect and low affinity for nontarget molecules,the antibody will likely be an acceptable reagent. Once the specificityof a binding molecule is established in one or more contexts, it may beemployed in other, preferably similar, contexts without necessarilyre-evaluating its specificity. Binding of two or more molecules may beconsidered specific if the affinity (equilibrium dissociation constant,Kd) is at least 10⁻³ M, preferably 10⁻⁴ M, more preferably 10⁻⁵ M, e.g.,10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, or 10⁻⁹ M under the conditions tested, e.g.,under physiological conditions.

“Significant sequence homology” as applied to an amino acid sequencemeans that the sequence displays at least approximately 20% identical orconservatively replaced amino acids, preferably at least approximately30%, at least approximately 40%, at least approximately 50%, at leastapproximately 60% identical or conservatively replaced amino acids,desirably at least approximately 70% identical or conservativelyreplaced amino acids, more desirably at least approximately 80%identical or conservatively replaced amino acids, and most desirably atleast approximately 90% amino acid identical or conservatively replacedamino acids relative to a reference sequence. When two or more sequencesare compared, any of them may be considered the reference sequence.Percent identity can be calculated using a FASTA or BLASTP algorithm,using default parameters. A PAM250 or BLOSUM62 matrix may be used. Forpurposes of calculating % identical or conservatively replaced residues,a conservatively replaced residue is considered identical to the residueit replaces. Conservative replacements may be defined in accordance withStryer, L., Biochemistry, 3rd ed., 1988, according to which amino acidsin the following groups possess similar features with respect to sidechain properties such as charge, hydrophobicity, aromaticity, etc. (1)Aliphatic side chains: G, A, V, L, I; (2) Aromatic side chains: F, Y, W;(3) Sulfur-containing side chains: C, M; (4) Aliphatic hydroxyl sidechains: S, T; (5) Basic side chains: K, R, H; (6) Acidic amino acids: D,E, N, Q; (7) Cyclic aliphatic side chain: P, which may be considered tofall within group (1).

“Subject”, as used herein, refers to an individual to whom an agent isto be delivered, e.g., for experimental, diagnostic, and/or therapeuticpurposes. Preferred subjects are mammals, particularly domesticatedmammals (e.g., dogs, cats, etc.), primates, or humans.

“Substantial sequence homology” as applied to a sequence means that thesequence displays at least approximately 60% identity, desirably atleast approximately 70% identity, more desirably at least approximately80% identity, and most desirably at least approximately 90% identityrelative to a reference sequence. When two or more sequences arecompared, any of them may be considered the reference sequence. %identity can be calculated using a FASTA, BLASTN, or BLASTP algorithm,depending on whether amino acid or nucleotide sequences are beingcompared. Default parameters may be used. A PAM250 or BLOSUM62 matrixmay be used.

“Supramolecular complex” refers to an assembly comprising at least twoentities that are physically associated with one another, in which oneor more entities is not covalently linked to another entity but isinstead associated with that entity by through one or morea nonspecificnoncovalent interactions mechanisms such as ionic interactions, hydrogenbonds, hydrophobic interactions, π-stacking, dative bonds, etc. Forexample, one or more entities may be entrapped, embedded, enclosed, orencapsulated within another entity, or entangled with another entity, ordissolved in another entity, or impregnated with another entity, oradsorbed to another entity, or bound to another entity, so as tomaintain a physical association between the entities. The entities maybe naturally occurring or synthetic. They may be, for example,polypeptides, non-polypeptide polymers, nucleic acids, lipids, smallmolecules, carbohydrates, etc. One or more of the entities may be arigid or flexible polymer scaffold, a three-dimensional structure suchas a microparticle, nanoparticle, liposome, dendrimer, etc. Thesupramolecular complex can contain any number or combination ofmolecules and/or other entities.

“Treating”, as used herein, refers to providing treatment, i.e.,providing any type of medical or surgical management of a subject. Thetreatment can be provided in order to reverse, alleviate, inhibit theprogression of, prevent or reduce the likelihood of a disease, disorder,or condition, or in order to reverse, alleviate, inhibit or prevent theprogression of, prevent or reduce the likelihood of one or more symptomsor manifestations of a disease, disorder or condition. “Prevent” refersto causing a disease, disorder, condition, or symptom or manifestationof such not to occur. Treating can include administering an agent to thesubject following the development of one or more symptoms ormanifestations indicative of a condition such as macular degeneration ordiabetic retinopathy, e.g., in order to reverse, alleviate, reduce theseverity of, and/or inhibit or prevent the progression of the conditionand/or to reverse, alleviate, reduce the severity of, and/or inhibit orone or more symptoms or manifestations of the condition. A compositionof this invention can be administered to a subject who has developed aneye disorder such as exudative or non-exudative ARMD or diabeticretinopathy or is at increased risk of developing such a disorderrelative to a member of the general population. A composition of thisinvention can be administered prophylactically, i.e., before developmentof any symptom or manifestation of the condition. Typically in this casethe subject will be at risk of developing the condition.

“Vector” is used herein to refer to a nucleic acid or a virus or portionthereof (e.g., a viral capsid) capable of mediating entry of, e.g.,transferring, transporting, etc., a nucleic acid molecule into a cell.Where the vector is a nucleic acid, the nucleic acid molecule to betransferred is generally linked to, e.g., inserted into, the vectornucleic acid molecule. A nucleic acid vector may include sequences thatdirect autonomous replication (e.g., an origin of replication), or mayinclude sequences sufficient to allow integration of part or all of thenucleic acid into host cell DNA. Useful nucleic acid vectors include,for example, DNA or RNA plasmids, cosmids, and naturally occurring ormodified viral genomes or portions thereof or nucleic acids (DNA or RNA)that can be packaged into viral capsids. Plasmid vectors typicallyinclude an origin of replication and one or more selectable markers.Plasmids may include part or all of a viral genome (e.g., a viralpromoter, enhancer, processing or packaging signals, etc.). Viruses orportions thereof (e.g., viral capsids) that can be used to introducenucleic acid molecules into cells are referred to as viral vectors.Useful viral vectors include adenoviruses, retroviruses, lentiviruses,vaccinia virus and other poxviruses, herpes simplex virus, and others.Viral vectors may or may not contain sufficient viral geneticinformation for production of infectious virus when introduced into hostcells, i.e., viral vectors may be replication-defective, and suchreplication-defective viral vectors may be preferable for therapeuticuse. Where sufficient information is lacking it may, but need not be,supplied by a host cell or by another vector introduced into the cell.The nucleic acid to be transferred may be incorporated into a naturallyoccurring or modified viral genome or a portion thereof or may bepresent within the virus or viral capsid as a separate nucleic acidmolecule. It will be appreciated that certain plasmid vectors thatinclude part or all of a viral genome, typically including viral geneticinformation sufficient to direct transcription of a nucleic acid thatcan be packaged into a viral capsid and/or sufficient to give rise to anucleic acid that can be integrated into the host cell genome and/or togive rise to infectious virus, are also sometimes referred to in the artas viral vectors. Where sufficient information is lacking it may, butneed not be, supplied by a host cell or by another vector introducedinto the cell.

Expression vectors are vectors that include regulatory sequence(s),e.g., expression control sequences such as a promoter, sufficient todirect transcription of an operably linked nucleic acid. An expressionvector comprises sufficient cis-acting elements for expression; otherelements for expression can be supplied by the host cell or in vitroexpression system. Such vectors typically include one or moreappropriately positioned sites for restriction enzymes, to facilitateintroduction of the nucleic acid to be expressed into the vector.

A “variant” of a particular polypeptide or polynucleotide has one ormore alterations (e.g., additions, substitutions, and/or deletions,which may be referred to collectively as “mutations”) with respect tothe polypeptide or nucleic acid, which may be referred to as the“original polypeptide or polynucleotide”. Thus a variant can be shorteror longer than the polypeptide or polynucleotide of which it is avariant. The terms “variant” encompasses “fragments”. A “fragment” is acontinuous portion of a polypeptide that is shorter than the originalpolypeptide. In certain embodiments of the invention a variantpolypeptide has significant sequence homology to the originalpolypeptide over a continuous portion of the variant that comprises atleast 50%, preferably at least 60%, at least 70%, at least 80%, at least90%, at least 95%, or more, of the length of the variant or the lengthof the polypeptide, (whichever is shorter). In certain embodiments ofthe invention a variant polypeptide has substantial sequence homology tothe original polypeptide over a continuous portion of the variant thatcomprises at least 50%, preferably at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or more, of the length of the variantor the length of the polypeptide, (whichever is shorter). In anon-limiting embodiment a variant has at least 80% identity to theoriginal sequence over a continuous portion of the variant thatcomprises between 90% and 100% of the variant, e.g., over 100% of thelength of the variant or the length of the polypeptide, (whichever isshorter). In another non-limiting embodiment a variant has at least 80%identity to the original sequence over a continuous portion of thevariant that comprises between 90% and 100% of the variant, e.g., over100% of the length of the variant or the length of the polypeptide,(whichever is shorter). In specific embodiments the sequence of avariant polypeptide has N amino acid differences with respect to anoriginal sequence, wherein N is any integer between 1 and 10. In otherspecific embodiments the sequence of a variant polypeptide has N aminoacid differences with respect to an original sequence, wherein N is anyinteger between 1 and 20. An amino acid “difference” refers to asubstitution, insertion, or deletion of an amino acid.

In certain embodiments of the invention a fragment or variant possessessufficient structural similarity to the original polypeptide so thatwhen its 3-dimensional structure (either actual or predicted structure)is superimposed on the structure of the original polypeptide, the volumeof overlap is at least 70%, preferably at least 80%, more preferably atleast 90% of the total volume of the structure of the originalpolypeptide. A partial or complete 3-dimensional structure of thefragment or variant may be determined by crystallizing the protein,which can be done using standard methods. Alternately, an NMR solutionstructure can be generated, also using standard methods. A modelingprogram such as MODELER (Sali, A. and Blundell, T L, J. Mol. Biol., 234,779-815, 1993), or any other modeling program, can be used to generate apredicted structure. If a structure or predicted structure of a relatedpolypeptide is available, the model can be based on that structure. ThePROSPECT-PSPP suite of programs can be used (Guo, J T, et al., NucleicAcids Res. 32(Web Server issue):W522-5, Jul. 1, 2004).

Preferably one, more than one, or all biological functions or activitiesof a variant or fragment is substantially similar to that of thecorresponding biological function or activity of the original molecule.For example, an activity of a variant or fragment is consideredsubstantially similar to the activity of the original molecule if theactivity of the variant or fragment is at least 20%, at least 50%, atleast 60%, at least 70%, at least 80%, or at least 90% of the activityof the original molecule, up to approximately 100%, approximately 125%,or approximately 150% of the activity of the original molecule. In othernonlimiting embodiments an activity of a variant or fragment isconsidered substantially similar to the activity of the originalmolecule if the amount or concentration of the variant needed to producean effect is within 0.5 to 5-fold of the amount or concentration of theoriginal molecule needed to produce that effect.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION Overview

The present invention provides compositions and methods for treatment ofeye disorders characterized by macular degeneration, choroidalneovascularization, retinal neovascularization, or any combination ofthe foregoing. The phrase “characterized by” is intended to indicatethat macular degeneration, CNV, and/or RNV, is a characteristic (i.e.,typical) feature of the disorder. Macular degeneration, CNV, and/or RNVmay be a defining and/or diagnostic feature of the disorder. Exemplarydisorders that are characterized by one or more of these features andcan be treated with the compositions and methods of the inventioninclude, but are not limited to, macular degeneration relatedconditions, diabetic retinopathy, and retinopathy of prematurity. Asmentioned above, macular degeneration refers to a variety ofdegenerative conditions characterized by central visual loss due todeterioration of the macula. By far the most common of these conditionsis age related macular degeneration (ARMD), which exists in both “dry”and “wet” forms. The invention provides a method of treating an eyedisorder characterized by macular degeneration, choroidalneovascularization, retinal neovascularization, or any combination ofthese, comprising (i) providing a subject in need of treatment for theeye disorder; and (ii) administering a composition comprising a viralcomplement control protein (VCCP) to the subject. The invention furtherprovides a method of treating an eye disorder characterized by maculardegeneration, choroidal neovascularization, retinal neovascularization,or any combination of these, comprising (i) providing a subject in needof treatment for the eye disorder; and (ii) administering a compositioncomprising a complement inhibiting fragment or variant of a VCCP to thesubject. The invention further provides a method of inhibiting CNV, RNV,or both, in the eye of a subject suffering from or at risk of an eyedisorder characterized by macular degeneration, choroidalneovascularization, retinal neovascularization, or any combination ofthese, comprising the step of: administering a composition comprising aVCCP to or in close proximity to the posterior segment of the subject'seye.

The events that occur in ARMD may be understood with reference to thevarious panels of FIG. 1. FIGS. 1A and 1B show structures present in theanterior and posterior segments of the eye, including the retina, whichcontains the macula. FIGS. 1C-1E depict the outer layers of a normal eye(1C), an eye suffering from dry ARMD (1D), and an eye suffering fromexudative (wet) ARMD (1E). The outer nuclear layer (ONL), containsnuclei of rod and cone photoreceptors. Each photoreceptor contains aninner segment (IS) and outer segment (OS), the latter of which containsthe pigment rhodopsin, which initiates the phototransduction cascadefollowing exposure to light. The retinal pigment epithelial layer (RPE)lies below the photoreceptors and above Bruch's membrane, a layer ofextracellular matrix that separates the RPE from a network ofcapillaries, the choriocapillaris (CC).

Dry ARMD is characterized by the existence of deposits known as drusenand the separation of the RPE from BM, which is accompanied by RPEatrophy and apoptosis and loss of underlying choriocapillaris andoverlying photoreceptors, resulting in areas of geographic atrophy whichcan eventually coalesce to form large patches. In exudative ARMD, newblood vessels grow from the choriocapillaris through Bruch's membraneand can extend into the RPE and photoreceptor cell layers (choroidalneovascularization). These blood vessels can bleed and leak fluid,frequently resulting in sudden visual loss due to events such as RPEand/or retinal detachment. Eventually a fibrovascular scar may form,leading to irreversible visual loss. In some forms of neovascular ARMD,angiomatous proliferation originates from the retina and extendsposteriorly into the subretinal space, eventually communicating in somecases with choroidal new vessels. This form of neovascular ARMD, termedretinal angiomatous proliferation (RAP), can be particularly severe. Ithas been suggested that angiomatous proliferation within the retina isthe first manifestation of the vasogenic process in this form ofneovascular ARMD. Dilated retinal vessels and pre-, intra-, andsubretinal hemorrhages and exudate evolve, surrounding the angiomatousproliferation as the process extends into the deep retina and subretinalspace.

The present invention provides compositions and methods that inhibit oneor more of the events or processes that takes place in ARMD. Theinvention is based at least in part on the discovery that certain viralproteins, e.g., viral complement control proteins (VCCPs) areparticularly suitable as therapeutic agents for macular degeneration andrelated conditions, for diabetic retinopathy, and for choroidal and/orretinal neovascularization due to these causes or others. For example,as described in Example 2, vaccinia virus complement control protein(VCP) was shown to be effective in significantly inhibiting thedevelopment of CNV in an animal model, i.e., VCP was effective inpreventing at least some of the CNV that would otherwise have occurred.To the best of the inventors' knowledge, this work represents the firstdemonstration that administration of an inhibitor of complementactivation to a subject is effective in inhibiting and at leastpartially preventing development of neovasculature in the eye and is thefirst demonstration that these agents will be effective treatments foreye disorders discussed herein.

Other viral proteins of use in the invention include, but are notlimited to, viral complement interfering proteins. To facilitateunderstanding of the invention, the complement system will first bebriefly outlined. Further information is found in the references citedherein. Subsequent sections describe the viral proteins of the presentinvention, compositions containing them, methods of use, etc.

Complement Pathways

The complement system plays a crucial role in a number of physiologicalprocesses including the response to injury and defense against foreignentities such as infectious agents. The complement system is also knownto play a role in a number of diseases (Makrides, S C, Pharm Rev.,50(1): 59-87). The complement system comprises more than 30 serum andcellular proteins that are involved in two major pathways, known as theclassical and alternative pathways (Kuby Immunology, 2000).

The classical pathway is usually triggered by binding of a complex ofantigen and IgM or IgG antibody to C1 (though certain other activatorscan also initiate the pathway). Activated C1 cleaves C4 and C2 toproduce C4a and C4b, in addition to C2a and C2b. C4b and C2a combine toform C3 convertase, which cleaves C3 to form C3a and C3b. Binding of C3bto C3 convertase produces C5 convertase, which cleaves C5 into C5a andC5b. C3a, C4a, and C5a are anaphylatoxins and mediate multiple reactionsin the acute inflammatory response. C3a and C5a are also chemotacticfactors that attract immune system cells such as neutrophils. C3 and C5convertase activity is controlled by a number of endogenous members ofthe Regulators of Complement Activation (RCA) family, also calledComplement Control Protein (CCP) family, which includes complementreceptor type 1 (CR1; C3b:C4b receptor), complement receptor type 2(CR2), membrane cofactor protein (MCP; CD46), decay-accelerating factor(DAF), factor H (fH), and C4b-binding protein (C4bp). Makrides, 1998,and references therein describe the complement system and itscomponents. RCA proteins are also described in U.S. Pat. No. 6,897,290.

The alternative pathway is initiated by microbial surfaces and variouscomplex polysaccharides. In this pathway, C3b, resulting from cleavageof C3, which occurs spontaneously at a low level, binds to targets oncell surfaces and forms a complex with factor B, which is later cleavedby factor D, resulting in a C3 convertase. Cleavage of C3 and binding ofanother molecule of C3b to the C3 convertase gives rise to a C5convertase. C3 and C5 convertases of this pathway are regulated by CR1,DAF, MCP, and fH. The mode of action of these proteins involves eitherdecay accelerating activity (i.e., ability to dissociate convertases),ability to serve as cofactors in the degradation of C3b or C4b by factorI, or both.

The C5 convertases produced in both pathways cleave C5 to produce C5aand C5b. C5b then binds to C6, C7, and C8 to form C5b-8, which catalyzespolymerization of C9 to form the C5b-9 membrane attack complex (MAC).The MAC inserts itself into target cell membranes and causes cell lysis.Small amounts of MAC on the membrane of cells may have a variety ofconsequences other than cell death.

A third complement pathway, the lectin complement pathway is initiatedby binding of mannose-binding lectin (MBL) and MBL-associated serineprotease (MASP) to carbohydrates. In the human lectin pathway, MASP-1and MASP-2 are involved in the proteolysis of C4, C2 and C3, leading toa C3 convertase described above.

As mentioned above, complement activity is regulated by variousmammalian proteins referred to as complement control proteins (CCPs).These proteins differ with respect to ligand specificity andmechanism(s) of complement inhibition (Lisczewski, M K and Atkinson, JP, in The Human Complement System in Health and Disease, eds. Volanakis,J E and Frank, M M, Dekker, New York, pp. 149-66, 1998). They mayaccelerate the normal decay of convertases and/or function as cofactorsfor factor I, to enzymatically cleave C3b and/or C4b into smallerfragments. CCPs are characterized by the presence of multiple (typically4-56) homologous motifs known as short consensus repeats (SCR),complement control protein (CCP) modules, or SUSHI domains (Reid, K B Mand Day, A J, Immunol Today, 10:177-80, 1989). These domains, consistingof approximately 50-70 amino acids, typically about 60 amino acids, arecharacterized by a conserved motif that includes four disulfide-bondedcysteines (two disulfide bonds), proline, tryptophan, and manyhydrophobic residues. FIG. 2 shows an SCR consensus sequence. It is tobe understood that any particular SCR may differ from the consensus atone or more positions.

Virus Complement Control Proteins and Methods of Use Thereof

The present invention provides a method of treating an eye disordercharacterized by macular degeneration, choroidal neovascularization,retinal neovascularization, or any combination of these, comprisingsteps of: (i) providing a patient at risk of or suffering from the eyedisorder; and (ii) administering a composition comprising a VCCP to thesubject. The invention further provides a method of treating an eyedisorder characterized by macular degeneration, choroidalneovascularization, retinal neovascularization, or any combination ofthese, comprising steps of: (i) providing a patient at risk of orsuffering from the eye disorder; and (ii) administering a compositioncomprising a complement inhibiting variant or fragment of a VCCP to thesubject. Viral complement control proteins (VCCPs) encoded by members ofthe poxvirus or herpesvirus families are of particular use in thepresent invention.

Poxviruses and herpesviruses are families of large, complex viruses witha linear double-stranded DNA genome. A number of these viruses infectanimals and can cause a range of diseases, the most feared of which inhumans is smallpox. Certain of these viruses encode a number ofimmunomodulatory proteins that are believed to play a role inpathogenesis by subverting one or more aspects of the normal immuneresponse and/or fostering development of a more favorable environment inthe host organism (Kotwal, G J, Immunology Today, 21(5), 242-248, 2000).Among these are viral complement control proteins. Poxvirus complementcontrol proteins are members of the complement control protein (CCP)superfamily and typically contain 4 SCR modules. The invention featuresthe discovery that these proteins possess features that make themparticularly advantageous for treatment and prevention of maculardegeneration related conditions and for treatment and prevention ofchoroidal neovascularization.

Thus in certain embodiments of the invention the VCCP is a poxviruscomplement control protein (PVCCP). The PVCCP can comprise a sequenceencoded by, e.g., vaccinia virus, variola major virus, variola minorvirus, cowpox virus, monkeypox virus, ectromelia virus, rabbitpox virus,myxoma virus, Yaba-like disease virus, or swinepox virus. In otherembodiments the VCCP is a herpesvirus complement control protein(HVCCP). The HVCCP can comprise a sequence encoded by a Macaca fuscatarhadinovirus, cercopithecine herpesvirus 17, or human herpes virus 8. Inother embodiments the HVCCP comprises a sequence encoded by herpessimplex virus saimiri ORF 4 or ORF 15 (Albrecht, J C. & Fleckenstein,B., J. Virol., 66, 3937-3940, 1992; Albrecht, J., et al., Virology, 190,527-530, 1992).

The VCCP may inhibit the classical complement pathway, the alternatecomplement pathway, the lectin pathway, or any combination of these. Incertain embodiments of the invention the VCCP, e.g., a PVCCP, binds toC3b, C4b, or both. In certain embodiments of the invention the PVCCPcomprises one or more putative heparin binding sites (K/R-X-K/R) and/orpossesses an overall positive charge. Preferably the PVCCP comprises atleast 3 SCR modules (e.g., modules 1-3), preferably 4 SCR modules. ThePVCCP protein can be a precursor of a mature PVCCP (i.e., can include asignal sequence that is normally cleaved off when the protein isexpressed in virus-infected cells) or can be a mature form (i.e.,lacking the signal sequence).

Vaccinia complement control protein (VCP), the first poxvirus complementcontrol protein to be identified, is a virus-encoded protein secretedfrom vaccinia infected cells. VCP is 244 amino acids in length, contains4 SCRs, and is naturally produced by intracellular cleavage of a 263amino acid precursor. VCP runs as an ˜35 kD protein in a 12%SDS/polyacrylamide gel under reducing conditions and has a predictedmolecular mass of about 28.6 kD. VCP is described in U.S. Pat. Nos.5,157,110 and 6,140,472, and in Kotwal, G K, et al., Nature, 355,176-178, 1988. FIGS. 3A and 3B show the sequence of the precursor andmature VCP proteins, respectively. VCP has been shown to inhibit theclassical pathway of complement activation via its ability to bind to C3and C4 and act as a cofactor for factor I mediated cleavage of thesecomponents as well as promoting decay of existing convertase (Kotwal, GK, et al., Science, 250, 827-830, 1990; McKenzie et al., J. Infect.Dis., 1566, 1245-1250, 1992). It has also been shown to inhibit thealternative pathway by causing cleavage of C3b into iC3b and therebypreventing the formation of the alternative pathway C3 convertase (Sahu,A, et al., J. Immunol., 160, 5596-5604, 1998). VCP thus blockscomplement activation at multiple steps and reduces levels of theproinflammatory chemotactic factors C3a, C4a, and C5a.

VCP also possesses the ability to strongly bind heparin in addition toheparan sulfate proteoglycans. VCP contains two putative heparin bindingsites located in modules 1 and 4 (Jha, P and Kotwal, G J, and referencestherein). VCP is able to bind to the surface of endothelial cells,possibly via interaction with heparin and/or heparan sulfate at the cellsurface, resulting in decreased antibody binding (Smith, S A, et al., J.Virol., 74(12), 5659-5666, 2000). VCP can be taken up by mast cells andpossibly persist in tissue for lengthy periods of time, therebypotentially prolonging its activity (Kotwal, G J, et al., In G P.Talwat, et al. (eds), 10^(th) International Congress of Immunology.,Monduzzi Editore, Bologna, Italy, 1998). In addition, VCP can reducechemotactic migration of leukocytes by blocking chemokine binding(Reynolds, D, et al., in S. Jameel and L. Villareal (ed., Advances inanimal virology. Oxford and IBN Publishing, New Delhi, India, 1999). VCPand other PVCCPs have a relatively small size relative to mammalianCCPs, which is advantageous for delivery. The crystal structure of VCPhas been determined (Murthy, K H M, et al., Cell, 104, 301-311, 2001).In addition, solution structures of various recombinantly producedproteins containing 2 or 3 SCRs from VCP have been determined.

Variola virus major and minor encode proteins that are highly homologousto VCP and are referred to as smallpox inhibitor of complement enzymes(SPICE) (Rosengard, A M, et al., Proc. Natl. Acad. Sci., 99(13),8803-8813. U.S. Pat. No. 6,551,595). SPICE from various variola strainssequenced to date differs from VCP by about 5% (e.g., about 11 aminoacid differences). Similarly to VCP, SPICE binds to C3b and C4b andcauses their degradation, acting as a cofactor for factor I. However,SPICE degrades C3b approximately 100 times as fast as VCP and degradesC4b approximately 6 times as fast as VCP. The amino acid sequence ofSPICE is presented in FIG. 6 (SEQ ID NO: 12) and can be described asfollows. Referring to FIG. 6, a signal sequence extends from amino acid1 to about amino acid 19. Four SCRs extend from about amino acid 20 toamino acid 263. Each SCR is characterized by four cysteine residues. Thefour cysteine residues form two disulfide bonds in the expressedprotein. The boundaries of each SCR are best defined by the first andfourth cysteine residues in the sequence that forms the disulfide bondsof the SCR. An invariant tryptophan residue is present between cysteine3 and cysteine 4 of each SCR. SCR1 extends from amino acid 20 or 21 toamino acid 81. Both residues are cysteines that may be involved indisulfide bonding. SCR2 extends from amino acid 86 to amino acid 143.SCR3 extends from amino acid 148 to amino acid 201. SCR4 extends fromamino acid 206 to amino acid 261. The SCRs include the complementbinding locations of SPICE. SPICE or any of the portions thereof thatinhibit complement activation, e.g., SPICE and SPICE-relatedpolypeptides containing four SCRs, such as those described in U.S. Pat.No. 6,551,595, are of use in the present invention.

Complement control proteins from cowpox virus (referred to asinflammation modulatory protein, IMP) and monkeypox virus (referred toherein as monkeypox virus complement control protein, MCP) have alsobeen identified and sequenced (Miller, C G, et al., Virology, 229,126-133, 1997 and Uvarova, E A and Shchelkunov, S N, Virus Res.,81(1-2), 39-45, 2001). MCP differs from the other PVCCPs describedherein in that it contains a truncation of the C-terminal portion of thefourth SCR.

It will be appreciated that the exact sequence of complement controlproteins identified in different virus isolates may differ slightly.Such proteins fall within the scope of the present invention. Complementcontrol proteins from any such isolate may be used, provided that theprotein has not undergone a mutation that substantially abolishes itsactivity. Thus the sequence of a VCCP such as SPICE or VCP may differfrom the exact sequences presented herein or under the accession numberslisted in Table 1. It will also be appreciated that a number of aminoacid alterations, e.g., additions, deletions, or substitutions such asconservative amino acid substitutions, may be made in a typicalpolypeptide such as a VCCP without significantly affecting its activity,such that the resulting protein is considered equivalent to the originalpolypeptide. For example, up to about 10% of the amino acids, or up toabout 20% of the amino acids may frequently be changed withoutsignificantly altering the activity. Also, of course, domains known tohave similar functions can be substituted for one another. Such domainsmay be found within a single polypeptide (e.g., repeated domains) orwithin different, homologous polypeptides. The effect of any particularamino acid alteration(s) or domain substitutions can readily bedetermined.

FIG. 4 shows a sequence alignment of a variety of poxvirus complementcontrol proteins from isolates of variola major and minor, vaccinia,cowpox virus, and monkeypox virus. FIG. 5 shows a comparison of the SCRdomain structure of a number of complement control proteins andfragments thereof, the number of K+R residues, %K+R residues, pI, numberof putative heparin binding sites, and ability to inhibit hemolysis(indicative of complement inhibiting activity) and/or bind to heparin.

Without limitation, any of the viral polypeptides identified byaccession number in Table 1 below is of use in various embodiments ofthe invention.

TABLE 1 Representative Viral Complement Control Proteins Virus ProteinAccession Virus Type Variola D12L NP_042056 Orthopoxvirus D15L (SPICE)AAA69423 Orthopoxvirus Vaccinia VCP AAO89304 Orthopoxvirus CowpoxCPXV034 AAM13481 Orthopoxvirus C17L CAA64102 Orthopoxvirus MonkeypoxD14L AAV84857 Orthopoxvirus Ectromelia virus Complement control CAE00484Orthopoxvirus protein Rabbitpox RPXV017 AAS49730 Orthopoxvirus Macacafuscata JM4 AAS99981 Rhadinavirus rhadinovirus (Herpesvirus)Cercopithecine Complement binding NP_570746 Herpesvirus herpesvirus 17protein (ORF4) Human herpes Complement binding AAB62602 Herpesvirusvirus 8 protein (ORF4)

Viral Complement Interfering Proteins

In addition to the VCCPs described above, there are a number of otherviral proteins that interfere with one or more steps in a complementpathway. These proteins are also of use in the present invention. Unlikethe VCCPs, certain of these proteins do not necessarily display clearhomology to cellular complement regulators. For example, HSV-1, HSV-2,VZV, PRV, BHV-1, EHV-1, and EHV-4 all encode versions of a conservedglycoprotein known as gC (Schreurs, et al., J. Virol., 62, 2251-2257,1988; Mettenleiter, et al, J. Virol., 64, 278-286; 1990; Herold, et al.,J. Virol., 65, 1090-1098; 1991). With the exception of VZV, the gCprotein encoded by these viruses binds to C3b (Friedman, et al., Nature,309, 633-634, 1984; Huemer, et al., Virus Res., 23, 271-280, 1993) gC1(from HSV-1) accelerates decay of the classical pathway C3 convertaseand inhibits binding of properdin and C5 to C3. Purified EBV virionspossess an activity that accelerates decay of the alternative pathway C3convertase and serves as a cofactor for the complement regulatoryprotein factor 1 (Mold et al., J Exp Med, 168, 949-969, 1988). Theforegoing proteins are referred to collectively as virus complementinterfering proteins (VCIPs). By any of a variety of means, such asinterfering with one or more steps of complement activation,accelerating decay of a complement component, and/or enhancing activityof a complement regulatory protein, these VCIPs are said to inhibitcomplement. Any of these proteins, or derivatives thereof, e.g.,fragments or variants thereof, can be used as a therapeutic agent in theinvention. As in the case of VCCPs, will be appreciated that the exactsequence of VCIPs identified in different virus isolates may differslightly. Such proteins fall within the scope of the present invention.In general, VCIPs may be used in a manner similar to the methodsdescribed herein for VCCPs, and wherever a method or compositioninvolving a VCCP is described, it should be understood that theinvention also provides similar methods involving a VCIP, even if notspecifically set forth herein, unless indicated otherwise.

Fragments and Variants of a VCCP or VCIP

In certain embodiments of the invention a fragment or variant of a VCCPor VCIP is administered to a subject to treat or prevent an eye disordercharacterized by macular degeneration, choroidal neovascularization,retinal neovascularization, or any combination of these. A fragment of aVCCP or VCIP contains fewer amino acids than the VCCP or VCIP while avariant may contain fewer, more, or the same number of amino acids asthe VCCP or VCIP. Preferred fragments and variants of a PVCCP possess atleast one of the following activities: (i) ability to bind to C3, C3b,or both; (ii) ability to act as a cofactor for factor I cleavage of C3;(iii) ability to bind to C4, C4b, or both; (iv) ability to act as acofactor for factor I cleavage of C4; (v) ability to accelerate decay ofexisting C3 convertase of the classical pathway, alternate pathway, orboth; (vi) ability to bind heparin; (vii) ability to bind to heparansulfate proteoglycans; (viii) ability to reduce chemotactic migration ofleukocytes; (ix) ability to block chemokine (e.g., MIP-1α) binding,e.g., to the surface of a cell (e.g., a leukocyte or endothelial cellsurface); (x) ability to inhibit antibody binding to class I MHCmolecules; (xi) ability to inhibit the classical complement pathway;(xii) ability to inhibit the alternative complement pathway; and (xiii)ability to inhibit complement-mediated cell lysis. Preferred PVCCPfragments and variants display complement binding activity, by which ismeant ability to detectably bind to one or more complement components,preferably selected from the group consisting of: C3, C3b, C4, and C4b.Preferred fragments or variants of HVCCPs may also display ability todetectably bind to one or more complement components. Preferably thebinding of the VCCP to the complement component is specific. It will beunderstood that a VCCP may be able to bind to only a single complementcomponent or may be able to bind to more than one different complementcomponent. Preferred VCCP fragments and variants are able to detectablyinhibit the classical complement pathway, alternate complement pathway,or both. Complement inhibiting or complement component binding activitycan be measured using any of a variety of methods known in the art.

Preferably a fragment or variant displaying any of the above activitiesdisplays such activity at a level at least 10%, at least 20%, at least30%, at least 40%, or at least 50% of the activity of VCP. In yet morepreferred embodiments of the invention the fragment or variant displaysat least 60%, at least 70%, at least 80%, at least 90%, or about 100% ofthe activity of VCP. In other preferred embodiments the fragment orvariant displays at least 10%, at least 20%, at least 30%, at least 40%,or at least 50% of the activity of SPICE. In yet more preferredembodiments of the invention the fragment or variant displays at least60%, at least 70%, at least 80%, at least 90%, or about 100% of theactivity of SPICE. In certain embodiments a fragment or variant displaysone or more activities at a greater level than VCP or at a greater levelthan SPICE. In certain embodiments of the invention a fragment orvariant displays an activity at a level at least 10%, at least 20%, atleast 30%, at least 40%, or at least 50% of the activity of SPICE. Inyet more preferred embodiments of the invention the fragment or variantdisplays at least 60%, at least 70%, at least 80%, at least 90%, orabout 100% of the activity of SPICE. In certain embodiments a fragmentor variant displays one or more activities at a greater level thanSPICE.

In general, fragments and variants of a VCIP fulfill similar criteria asthose described above for fragments or variants of a VCCP, except thatthe relevant activity is complement interfering activity or complementcomponent binding activity of the naturally occurring VCIP.

Certain variants of a naturally occurring VCCP or VCIP contain one ormore conservative amino acid substitutions with respect to the naturallyoccurring form. Certain variants of a naturally occurring VCCP containonly conservative amino acid substitutions with respect to the naturallyoccurring form.

The invention encompasses the use of VCCPs or VCIPs discussed hereinthat differ from their naturally occurring counterparts by one or moreamino acid substitutions, additions, or deletions. Each amino acid addedto, deleted from, or altered in the naturally occurring amino acidsequence with respect to the naturally occurring sequence is consideredto constitute an amino acid difference. The minimum number of suchadditions, deletions, or alterations that must be performed to arrive atthe fragment or variant is considered to be the number of differences.In certain embodiments of the invention a VCCP or VCIP contains 1, 2, 3,4, or 5 amino acid differences, 5-10 amino acid differences, 10-15 aminoacid differences, 15-25 amino acid differences, 25-50 amino aciddifferences, or 50-100 amino acid differences with respect to itsnaturally occurring counterpart. In certain embodiments of the inventionthe number of amino acid differences between a naturally occurring VCCPor VCIP and a fragment or variant thereof for use in the invention is 5%or less, 10% or less, or 25% or less of the total number of amino acidsin the naturally occurring protein. Preferred fragments of a VCCPcomprise at least 3 SCR modules, preferably 4 SCR modules.

In certain embodiments of the invention a fragment or variant of anaturally occurring VCCP is at least 20% identical, at least 30%identical, at least 40% identical, at least 50% identical, at least 60%identical, at least 70% identical, at least 80% identical, at least 90%identical, at least 95% identical to its naturally occurringcounterpart, over one or more SCR modules, e.g., 1, 2, 3, or 4 SCRmodules. The amino acid portion is preferably at least 20 amino acids inlength, more preferably at least 50 amino acids in length.

In certain embodiments of the invention a fragment or variant displayssignificant sequence homology to a naturally occurring PVCCP encoded bya vaccinia virus, variola major virus, variola minor virus, cowpoxvirus, or monkeypox virus. Preferably the fragment or variant possessesability to bind to one or more complement components. Preferably thefragment or variant displays substantial sequence homology to anaturally occurring PVCCP over the length of the fragment or variant.The PVCCP fragment or variant may inhibit the classical complementpathway, the alternate complement pathway, or both. In certainembodiments of the invention the PVCCP fragment or variant binds to C3b,C4b, or both. In certain embodiments of the invention the PVCCP fragmentor variant comprises one or more putative heparin binding sites(K/R-X-K/R) and/or possesses an overall positive charge.

In preferred embodiments of the invention the PVCCP fragment or variantcomprises at least 3 SCR modules (e.g., modules 1-3), preferably 4 SCRmodules. Preferably each of the SCR modules displays significantsequence homology or, more preferably, substantial sequence homology, toan SCR module found in a naturally occurring PVCCP, e.g., VCP or SPICE.Preferably the multiple SCR modules are arranged in an N to C manner soas to maximize overall identity to a naturally occurring PVCCP. If thesequence of a PVCCP fragment or variant contains an SCR domain thatdiffers from the SCR consensus sequence at one or more positions, incertain embodiments of the invention the amino acid(s) at the one ormore differing positions is identical to that found at a correspondingposition in the most closely related SCR found in a naturally occurringPVCCP. In certain embodiments the PVCCP variant comprises at least oneSCR module from a first PVCPP and at least one SCR module from a secondPVCPP. In certain embodiments the PVCCP variant comprises at least oneSCR module from a PVCCP and at least one SCR from a mammalian complementcontrol protein (RCA protein). Any number of SCR modules, e.g., 1, 2, 3,4, or more can come from any particular PVCCP or RCA protein in variousembodiments of the invention. All such combinations and permutations arecontemplated, even if not explicitly set forth herein.

Generally a fragment or variant of a naturally occurring VCCP or VCIPpossesses sufficient structural similarity to its naturally occurringcounterpart that it is recognized by a polyclonal antibody thatrecognizes the naturally occurring counterpart. In certain embodimentsof the invention a fragment or variant of a VCCP possesses sufficientstructural similarity to VCP or SPICE so that when its 3-dimensionalstructure (either actual or predicted structure) is superimposed on thestructure of VCP or SPICE, the volume of overlap is at least 70%,preferably at least 80%, more preferably at least 90% of the totalvolume of the VCP structure. A partial or complete 3-dimensionalstructure of the fragment or variant may be determined by crystallizingthe protein as described for VCP (Murthy, 2001). Alternately, an NMRsolution structure can be generated, which has been performed forvarious VCP fragments (Wiles, A P, et al., J. Mol. Biol. 272, 253-265,1997). A modeling program such as MODELER (Sali, A. and Blundell, T L,J. Mol. Biol., 234, 779-815, 1993), or any other modeling program, canbe used to generate a predicted structure. The model can be based on theVCP structure and/or any known SCR structure. The PROSPECT-PSPP suite ofprograms can be used (Guo, J T, et al., Nucleic Acids Res. 32(Web Serverissue):W522-5, Jul. 1, 2004). Similar methods may be used to generate astructure for SPICE.

Fragments or variants of a VCCP or VCIP may be generated by anyavailable means, a large number of which are known in the art. Forexample, VCCPs, VCIPs, and fragments or variants thereof can be producedusing recombinant DNA technology as described below. Sequences for aVCCP or VCIP fragment may be chemically synthesized, produced using PCRamplification from a cloned VCCP or VCIP sequence, generated by arestriction digest, etc. Sequences for a VCCP variant may be generatedby random mutagenesis of a VCCP sequence (e.g., using X-rays, chemicalagents, or PCR-based mutagenesis), site-directed mutagenesis (e.g.,using PCR or oligonucleotide-directed mutagenesis, etc. Selected aminoacids can be changed or added.

While not wishing to be bound by any theory, it is likely that aminoacid differences between naturally occurring PVCCPs occur at positionsthat are relevant in conferring differences in particular propertiessuch as ability to bind heparin, activity level, etc. For example, VCPand SPICE differ at only 11 amino acids, but SPICE has a much higheractivity as a cofactor for cleavage of C3b (e.g., cleavage occurs at amuch faster rate with SPICE than with VCP). The amino acid differencesare likely to be responsible for the differential activities of the twoproteins. The amino acids at these positions are attractive candidatesfor alteration to identify variants that have yet greater activity.

Any variant or fragment may be tested as described below, e.g., using invitro assays, virus-based assays, and/or by administering the variant orfragment to an animal that constitutes an experimental model for an eyedisorder such as a macular degeneration related condition or diabeticretinopathy, e.g., an animal exhibiting choroidal neovascularization. Avariant or fragment that treats or prevents one or more features ofmacular degeneration, diabetic retinopathy, and/or choroidalneovascularization in the animal is identified as a potentially usefultherapeutic agent. Such agents may be further tested in other animalmodel systems and/or in human subjects.

It should be understood that the invention includes embodiments in whichany of the methods of the invention in which a VCCP or VCIP is used arepracticed using one or more effective fragments or variants of the VCCPor VCIP.

Producing a VCCP, VCIP, or a Fragment or Variant Thereof

A VCCP, VCIP, or a fragment or variant of either may be produced usingany suitable method known in the art. For purposes of brevity, thefollowing discussion refers to VCCPs, but it will be understood that thesame methods can also be applied in the case of VCIPs. According to oneapproach, appropriate host cells (e.g., RK-13, L cells, etc.) can beinfected with a virus that encodes a VCCP. The cells are maintained inculture and secrete VCCP into the medium. Medium is harvested, and theVCCP is purified therefrom using standard techniques such asconcentration and column chromatography. U.S. Pat. No. 5,157,110describes methods for obtaining a substantially pure preparation of VCPfrom virus-infected cells. Immunoaffinity chromatography with antibodiesthat specifically bind to the VCCP can be used. While not wishing to bebound by any theory, the relatively small size of certain VCCP such asPVCCPs (compared, for example, to a number of mammalian CCPs),facilitates their expression and purification.

Recombinant DNA technology can be used to produce a VCCP or a VCCPfragment or variant according to methods that are well known in the art.Suitable methods for producing any polypeptide of choice usingrecombinant DNA technology are described in U.S. Pub. No. 20030219406and in numerous standard reference works. Briefly, a polynucleotide thatencodes the VCCP or VCCP fragment or variant is obtained. Thepolynucleotide can be obtained, e.g., by cloning or PCR from virus orvirus-infected cells. Polynucleotides encoding VCCPs have already beenisolated and are available. Furthermore, a number of poxvirus andherpesvirus genomes encoding VCCPs have been completely sequenced. Thepolynucleotide is inserted into a suitable expression vector to generatea recombinant polynucleotide. The expression vector is selected suchthat it can direct expression of the recombinant polynucleotide in ahost cell, resulting in production of a recombinant polypeptide. Thusonce inserted into the expression vector, the coding sequence for thepolynucleotide should be located in suitable proximity to appropriateexpression control elements, e.g., regulatory elements such as apromoter, initiation signals, termination signals, polyadenylationsignals, etc., depending upon the particular host cell into which theexpression vector is to be introduced. One of ordinary skill in the artwill be able to select appropriate expression control elements.

Numerous expression vectors suitable for use in prokaryotic oreukaryotic host cells are known in the art. Either plasmid or viralvectors can be used. For example, for expression in E. coli the pALTER™or PinPoint™ vectors (Promega) can be used. For expression in S.cerevisiae pYES vectors, which contain a GAL promoter to driveexpression, can be used. For expression in Pichia pastoris the pHIL-S1vector can be used. Pichia pastoris expression systems have been usedfor expression of high levels of recombinant VCP and fragments thereof(Sahu, 1998). For expression in insect cells, a coding sequence can beinserted into a transfer vector such as pFastBac™ or pPolh(Sigma-Aldrich), which is then used to create a recombinant baculovirusthat is used to infect insect cells. For expression in plant cells, aplant virus vector such as an alfalfa mosaic virus or tobacco mosaicvirus vector, or Agrobacterium tumefaciens mediated gene transfer can beused. Transgenic plants or animals expressing the protein can begenerated and used as a source. Methods for producing transgenic plantsand animals are well known in the art.

For expression in mammalian cells, numerous vectors containing promotersfunctional in mammalian cells, such as the cytomegalovirus (CMV)promoter, herpes simplex virus (HSV) promoter, SV40 promoters,retroviral LTR, etc. are available. The promoter can be inducible orconstitutive. In certain embodiments the expression vector comprisessites that permit homologous recombination into a host cell genome.

In certain embodiments of the invention the recombinant polypeptidecomprises a signal sequence, also referred to as a signal peptide, sothat the polypeptide is secreted. A variety of signal sequences areknown in the art. Signal sequences are typically characterized by a coreof hydrophobic amino acids which are generally cleaved from the matureprotein during secretion in one or more cleavage events. Such signalpeptides may contain processing sites that allow cleavage of the signalsequence from the mature proteins as they pass through the secretorypathway. Optionally any signal peptide present in the native VCCP orVCIP is removed and replaced with a signal peptide sequence from anotherpolypeptide. In certain embodiments of the invention the recombinantpolypeptide comprises a portion encoding a tag, e.g., a metal affinitytag (MAT) such as a 6X-His tag, HA tag, Myc tag, FLAG tag, GST tag, etc.Such tags may simplify the detection, isolation and/or purification ofthe protein. The recombinant polypeptide may comprise a cleavage site,which may be encoded by the inserted polynucleotide or by the vector.For example, the vector may comprise a portion that encodes a cleavagesite upstream or downstream of a portion that encodes a tag, so thatwhen a polynucleotide that encodes a VCCP or a VCCP fragment or variantis inserted between the portion that encodes the tag and the portionthat encodes a cleavage site, the resulting open reading frame encodes afusion protein containing a portion that comprises VCCP or a VCCPfragment or variant and a cleavage site. Cleavage of the fusion proteinat the cleavage site releases the encoded VCCP or VCCP fragment orvariant. The cleavage site may be a site for cleavage by chemical means(e.g., cyanogen bromide) or by enzymatic means (e.g., by a protease suchas trypsin, chymotrypsin, thrombin, pepsin, Staphylococcus aureus V8protease, and Factor Xa protease). Including a cleavage site allows thetag to be readily be removed from the translated polypeptide, ifdesired. Sequences encoding a signal sequence, tag and/or cleavage sitemay be present within a vector into which a particular polynucleotide isinserted and need not be present within the inserted polynucleotideitself. Members of the pFLAG™ and pMAT™ series of vectors(Sigma-Aldrich) can be used for expression of proteins with a FLAG orMAT tag in prokaryotic or mammalian cells.

The expression vector is introduced into an appropriate host cell usingany of a variety of methods known in the art including calcium-mediatedtransformation, electroporation, calcium-phosphate transfection,cationic lipid-mediated transfection, microparticle bombardment, etc.Cells that have taken up the expression vector are typically selected bygrowth in or on a selective medium. A stable cell line can be generated.Alternately, transient transfection can be used. The cells aremaintained in culture for a period of time to allow production of therecombinant polypeptide. Cells and/or medium are then harvested, and thepolypeptide is purified. The invention thus provides polynucleotides,expression vectors, and host cells, that encode a VCCP or fragment orvariant thereof and also polynucleotides, expression vectors, and hostcells that encode a polypeptide containing a portion that comprises aVCCP or fragment or variant thereof and portion that binds to a cell ornoncellular molecular entity (i.e., a binding moiety).

See, e.g., Hardin, C., et al., (Eds.), “Cloning, Gene Expression andProtein Purification: Experimental Procedures and Process Rationale”,Oxford University Press, Oxford, 2001, for further information regardingproduction of recombinant polypeptides and purification of polypeptides.

In certain embodiments of the invention rather than administering aVCCP, VCCP fragment or variant, or a polypeptide comprising a VCCP or aVCCP fragment or variant and a binding moiety that binds to a componentpresent on or at the surface of a cell or noncellular molecular entityto a subject, recombinant cells that produce the polypeptide areadministered. Such cells may be generated similarly to the recombinanthost cells used for protein expression (i.e., by introduction of anucleic acid such as an expression vector that encodes the VCCP or VCCPfragment or variant into the cell). Preferably a stable cell line isgenerated. The cells may be, for example, neural stem cells, RPE stemcells, RPE cells, etc. These cells may help reconstitute damaged RPEand/or photoreceptors. In other embodiments of the invention any othercell type may be used. Autologous cells may be used. The cells can beintroduced into the vitreous cavity or elsewhere.

Assessing Properties of a VCCP or VCIP or a Fragment or Variant Thereof

Any suitable method can be used for assessing any of the properties of aVCCP, VCIP, or a fragment or variant thereof, and such determinationrequires no more than routine experimentation. A number of in vitroassays can be used. For example, ability of an agent to inhibit theclassical or alternative complement pathway may be assessed by measuringcomplement-mediated hemolysis of erythrocytes (e.g., antibody-sensitizedor unsensitized rabbit or sheep erythrocytes), by human serum or a setof complement components in the presence or absence of the agent. Anagent inhibits complement if it decreases hemolysis in this inhibitionassay to a statistically significant degree (p>0.05). The IC₅₀ ofrecombinantly produced VCP for inhibition of the classical andalternative pathways (i.e., concentration required for 50% inhibition)has been measured (Sahu, 1998). In certain embodiments of the inventiona VCCP or VCCP fragment or variant has an IC₅₀ that is less than 5 timesthe IC₅₀ of VCP (either recombinantly produced VCP or VCP purified fromvirus-infected cells can be used). Preferably the fragment or varianthas an IC₅₀ that is less than 4 times, less than 3 times, less than 2times that of VCP. Preferably the IC₅₀ is approximately equal to that ofVCP (e.g., within 10% of the value for VCP), or even less than that ofVCP. In certain embodiments of the invention a VCCP or VCCP fragment orvariant has an IC₅₀ that is less than 5 times the IC₅₀ of SPICE (eitherrecombinantly produced SPICE or SPICE purified from virus-infected cellscan be used). Preferably the fragment or variant has an IC₅₀ that isless than 4 times, less than 3 times, less than 2 times that of SPICE.Preferably the IC₅₀ is approximately equal to that of SPICE (e.g.,within 10% of the value for SPICE), or even less than that of SPICE.

The ability of an agent to bind to one or more complement components canbe assessed using an ELISA assay. For example, the wells of a microtiterplate are coated with the agent. Complement component(s) are added tothe wells. After a period of incubation the wells are washed, and boundcomplement components are detected using antibodies. A direct ELISAassay has been used to quantitatively measure the ability ofrecombinantly produced VCP to bind to C3 and fragments thereof (Sahu,1998). Preferably the dissociation constant (K_(d)) of a VCCP or VCCPfragment or variant with respect to one or more complement components(e.g., C3, C3b, and/or C4b) is less than 100 times the K_(d) of VCP withrespect to the same component, preferably less than 10 times the K_(d),more preferably less than 5 times or less than 2 times. Preferably theK_(d) is approximately equal to that of VCP, or even lower than that ofVCP. In certain embodiments of the invention the dissociation constant(K_(d)) of a VCCP or VCCP fragment or variant with respect to one ormore complement components (e.g., C3, C3b, and/or C4b) is less than 100times the K_(d) of SPICE with respect to the same component, preferablyless than 10 times the K_(d), more preferably less than 5 times or lessthan 2 times. Preferably the K_(d) is approximately equal to that ofSPICE, or even lower than that of SPICE.

The ability of a VCCP fragment or variant, e.g., a PVCCP or PVCCPfragment or variant, to act as a cofactor for factor I mediated cleavageof a complement component, e.g., C3, C3b, etc., and the rate of suchcleavage, may be determined by incubating the agent with the complementcomponent and factor I for a period of time. Following incubationsamples are subjected to electrophoresis to separate the components andcleavage products by size. Complement components and cleavage productsthereof may be visualized using, for example, Coomassie staining,immunoblotting using antibodies that recognize the component, etc. Atime course may be performed. Preferably the rate is more than 0.1 timesthe rate of VCP-activated cleavage, more preferably more than 0.5 timesthe rate of VCP-activated cleavage, yet more preferably approximatelyequal to the rate of VCP-activated cleavage, or even greater. In certainembodiments of the invention the rate is more than 0.1 times the rate ofSPICE-activated cleavage, more preferably more than 0.5 times the rateof SPICE-activated cleavage, yet more preferably approximately equal tothe rate of SPICE-activated cleavage, or even greater.

The ability of an agent to bind heparin may be assessed by ELISA assayor by flowing the agent through a heparin column and collecting andanalyzing unbound material for presence of the agent (where a diminishedamount of the agent indicates that the agent has bound to heparin in thecolumn) Methods for assessing the ability of an agent to bind to cells,e.g., endothelial cells, include flow cytometry (Smith, 2000).Chemotaxis inhibition by an agent or cellular uptake of an agent can bemeasured using well established chemotaxis or uptake assays. In any ofthe above methods, the agent may be tested at a range of differentdilutions. In certain embodiments of the invention a PVCCP or PVCCPfragment or variant displays one or more of these activities at a levelup to 10-fold lower than that of VCP or SPICE, more preferably up to5-fold lower than that of VCP or SPICE, more preferably at approximatelythe same level as that of VCP or SPICE, or at an even greater level.

Virus-based assays can also be used. For example, it is known thatexpression of VCP inhibits antibody-dependent neutralization of vacciniavirus and virus-infected cells, and reduces cellular influx andinflammation. Deletion of the VCP gene reduces pathogenicity. Theactivity of a VCCP or VCCP fragment or variant can be tested byintroducing a virus that expresses the molecule (e.g., a recombinantvirus) into appropriate host cells and/or animals and measuring any oneor more of these parameters.

The foregoing methods are described in a number of references citedherein (U.S. Pat. No. 5,157,110 or 6,551,595; Sahu, 1998; Smith, 2000;Rosengard, 2002, etc.). Any of these methods or variants thereof, orothers known in the art, can be used.

Targeting VCCPs, VCIPs, or Fragments of Variants Thereof

The invention provides a composition comprising (i) a VCCP, a VCIP, or acomplement inhibiting fragment or variant of either; and (ii) a bindingmoiety that binds to a component present in the eye of a subject at riskof or suffering from an eye disorder characterized by maculardegeneration, choroidal neovascularization, retinal neovascularization,or any combination of these, e.g., a macular degeneration relatedcondition, diabetic retinopathy, or retinopathy of prematurity. Thecomposition can be used to treat or prevent any of the foregoingdisorders. Preferably the binding moiety and the VCCP, VCIP, or fragmentor variant of either, are linked. The linkage can be covalent ornoncovalent and can be direct or indirect in various embodiments of theinvention. The binding moiety can be, for example, an antibody orligand, as discussed below. According to certain embodiments of theinvention the component is a cellular marker. In other embodiments ofthe invention the component is a drusen constituent. The cellular markercan be any marker that is expressed on or at the surface of a cell,preferably an endothelial cell or retinal pigment epithelial cell. Incertain embodiments of the invention the cellular marker is a cell typespecific marker. For purposes of brevity, the discussion below refersprimarily to VCCPs. However, the invention includes embodiments in whicha VCCP fragment or variant or a VCIP or a VCIP fragment or variant isused.

In general, the component can be any molecule present on or at thesurface of a cell or noncellular molecular entity. By “on or at thesurface of the cell or noncellular molecular entity” is meant that thecomponent is accessible to molecules present in the extracellularenvironment so that it can be recognized and bound by the moiety. Thecomponent may be entirely extracellular. The component may be insertedinto the cell membrane. In certain embodiments of the invention thecomponent may be partly or entirely within the membrane, in which casethe entity must partially penetrate the membrane to gain access. Ingeneral, the component is not located in the cytoplasm of a cell. Aslong as a sufficient portion of the component is exposed or accessibleso that it can be recognized and bound, it will be said to be present onor at the surface. In preferred embodiments of the invention thecomponent is a cellular marker, e.g., a cell type specific marker. Wherethe target is a molecular entity other than a cell, the component can beany chemical entity present on or at the surface of the molecule that isrecognizable by an antibody or ligand.

A number of cellular markers that are expressed on or at the surface ofendothelial cells and can be used to target a VCCP to endothelial cellsin the eye (e.g., in the choroidal vasculature) are disclosed in U.S.Ser. No. 10/923,940. Tissue factor (TF), a molecule involved inhemostasis, is a preferred marker. Briefly, tissue factor is a cellmembrane-bound glycoprotein (MW 46 kDa) and a member of the class 2cytokine receptor family. It is composed of a hydrophilic extracellulardomain, a membrane-spanning hydrophobic domain, and a cytoplasmic tailof 21 residues, including a non-disulfide-linked cysteine. Upon exposureto blood, perivascular cell-bound TF binds to factor VII (FVII), avitamin K-dependent serine protease. TF is expressed on endothelialcells lining the luminal surface of various forms of pathologicalneovasculature, including pathological vasculature associated with theexudative (wet) form of age-related macular degeneration and diabeticretinopathy but typically is not expressed (or is expressed at a muchlower level) in normal vasculature, thus providing a specific andaccessible target. By linking a VCCP or VCIP to factor VII or aderivative thereof, the VCCP is targeted to cells that express TF, e.g.,endothelial cells in pathological neovasculature. Integrinalpha(v)beta(3) is another preferred marker.

A number of markers are expressed on or at the surface of retinalpigment epithelial cells. These include, but are not limited to, CD68antigen (Elner S G, Exp Eye Res. 1992 July; 55(1):21-8), claudin(Nishiyama K, et al., Anal Rec. 2002 Jul. 1; 267(3):196-203, the proteinencoded by the RPE65 gene (Nicoletti A., et al., Invest Ophthalmol VisSci. 1998 March; 39(3):637-44), CD45 and ICAM-1 (Limb, G A, et al., CurrEye Res. 1997 October; 16(10):985-91). See also Chowers, I., et al.,Studies on retinal and retinal pigment epithelial gene expression,Novartis Found Symp. 2004; 255:131-45, 145-6, 177-8 for additionalexamples.

A large number of molecular components have been identified in drusen.Such components are suitable noncellular molecular entities to which aVCCP of the present invention can be targeted. These constituentsinclude α1-antichymotrypsin, α1-antitrypsin, Alzheimer amyloid βpeptide, advanced glycation end products, amyloid β component,apolipoproteins B and E, carbohydrate moieties recognized by variouslectins, cholesterol esters, clusterin, complement factors, clusterdifferentiation antigen, complement receptor 1, factor X, heparansulfate proteoglycan, human leukocyte antigen DR, immunoglobulin lightchains, major histocompatibility complex class II antigens, membranecofactor protein, peroxidized lipids, phospholipids and neutral lipids,tissue inhibitor of matrix metalloproteinases-3, transthyretin,ubiquitin, and vitronectin (Zarbin, M A, Arch Ophthalmol. 122:598-614,2004). A number of these components are also found in depositsassociated with a variety of different diseases includingatherosclerosis.

In certain preferred embodiments of the invention the binding moiety islinked to a VCCP. In other embodiments the binding moiety comprises aportion that binds to another molecule to which a VCCP is attached.Suitable binding moieties include antibodies that specifically bind to acellular marker or noncellular molecular entity such as a drusenconstituent and ligands that specifically bind to a cellular marker ornoncellular molecular entity such as a drusen constituent. In general,the linkage between the binding moiety and the VCCP can be covalent ornoncovalent and can be direct or indirect in various embodiments of theinvention. Similarly, a moiety that binds to a noncellular marker suchas a drusen constituent may be linked to a VCCP or to another moleculeto which a VCCP is attached.

In those embodiments of the invention in which the binding moiety is anantibody, the antibody may be any immunoglobulin or a derivative thereofwhich maintains binding ability, or any protein having a binding domainwhich is homologous or largely homologous to an immunoglobulin bindingdomain. Such proteins may be derived from natural sources, or partly orwholly synthetically produced (e.g., using recombinant DNA techniques,chemical synthesis, etc.). The antibody can be of any species, e.g.,human, rodent, rabbit, goat, chicken, etc. The antibody may be a memberof any immunoglobulin class, including any of the human classes: IgG,IgM, IgA, IgD, and IgE. In various embodiments of the invention theantibody may be a fragment of an antibody such as an Fab′, F(ab′)₂, scFv(single-chain variable) or other fragment that retains an antigenbinding site, or a recombinantly produced scFv fragment, includingrecombinantly produced fragments. See, e.g., Allen, T., Nature ReviewsCancer, Vol. 2, 750-765, 2002, and references therein. Monovalent,bivalent or multivalent antibodies can be used. The antibody may be achimeric or “humanized” antibody in which, for example, a variabledomain of rodent origin is fused to a constant domain of human origin,thus retaining the specificity of the rodent antibody. It is noted thatthe domain of human origin need not originate directly from a human inthe sense that it is first synthesized in a human being. Instead,“human” domains may be generated in rodents whose genome incorporateshuman immunoglobulin genes. See, e.g., Vaughan, et al., (1998), NatureBiotechnology, 16:535-539. The antibody may be partially or completelyhumanized. An antibody may be polyclonal or monoclonal, though forpurposes of the present invention monoclonal antibodies are generallypreferred. Preferably the antibody specifically binds to its target onthe cell surface, e.g., to a cell-type specific marker. Methods forproducing antibodies that specifically bind to virtually any molecule ofinterest are known in the art. For example, monoclonal or polyclonalantibodies can be purified from natural sources, e.g., from blood orascites fluid of an animal that produces the antibody (e.g., followingimmunization with the molecule or an antigenic fragment thereof) or canbe produced recombinantly, in cell culture.

In certain embodiments of the invention it is preferable to use F(ab′)2or F(ab′) fragments rather than antibodies that contain an Fc portionsince the Fc portion may have a pro-inflammatory effect or cause otherundesirable effects. However, in certain embodiments of the invention itis preferred to use antibodies comprising an Fc domain. F(ab′)₂fragments can be generated, for example, through the use of anImmunopure F(ab′)₂ Preparation Kit (Pierce) in which the antibodies aredigested using immobilized pepsin and purified over an immobilizedProtein A column. The digestion conditions (such as temperature andduration) may be optimized by one of ordinary skill in the art to obtaina good yield of F(ab′)₂. The yield of F(ab′)₂ resulting from thedigestion can be monitored by standard protein gel electrophoresis.F(ab′) can be obtained by papain digestion of antibodies, or by reducingthe S-S bond in the F(ab′)₂.

In various embodiments of the invention an appropriate binding moiety towhich a VCCP is linked can be any molecule that specifically binds to atarget molecule (e.g., polypeptide or a portion thereof such as acarbohydrate moiety), through a mechanism other than an antigen-antibodyinteraction. Such a binding moiety is referred to as a “ligand”. Forexample, in various embodiments of the invention a ligand can be apolypeptide, peptide, nucleic acid (e.g., DNA or RNA), carbohydrate,lipid or phospholipid, or small molecule (e.g., an organic compound,whether naturally-occurring or artificially created that has relativelylow molecular weight and is not a protein, polypeptide, nucleic acid, orlipid, typically with a molecular weight of less than about 1500 g/moland typically having multiple carbon-carbon bonds).

Ligands may be naturally occurring or synthesized, including moleculeswhose structure has been invented by man. Examples of ligands include,but are not limited to, hormones, growth factors, or neurotransmittersthat bind to particular receptors. For example, Factor VII is a ligandfor TF. Exemplary TF binding moieties are FVII, activated FVII (FVIIa),inactive FVIIa, antibodies that bind to tissue factor, engineeredpolypeptides, aptamers, and small molecules that bind to tissue factor.Inactive FVII or inactive FVIIa is a derivative of FVII or FVIIa that iscatalytically inactivated in the active site, e.g., by derivatizationwith an inhibitor. Many irreversible serine protease inhibitors, whichgenerally form covalent bonds with the protease active site, are knownin the art. Examples of suitable inhibitors include peptide halomethylketones, e.g., peptide chloromethyl ketones (see, Williams et al., J.Biol. Chem. 264:7536-7540, 1989 and U.S. Pat. No. 5,817,788). In someembodiments FVII or FVIIa activity is inhibited by substitution,deletion, and/or insertion of one or more amino acids in FVII. Generallythe substitution(s), insertion(s), and/or deletion(s) are made at oradjacent to a catalytic site residue. In certain embodiments, thealteration(s) is a substitution or deletion of Ser344, Asp242, and/orHis193. As mentioned above, TF binds to factor VII that is normallypresent in the blood. Thus according to one embodiment of the inventiona VCCP is linked to a TF binding moiety. The binding moiety binds to TF,present on endothelial cells in choroidal neovasculature, therebyproviding an increased amount of the VCCP at the cell surface andpreventing additional complement activation.

It will also be appreciated that fragments or variants of theabove-mentioned polypeptide ligands differing in sequence from theirnaturally occurring counterparts but retaining the ability to bind toendothelial cells or retinal pigment epithelial cells can also be used.In certain embodiments of the invention a polypeptide ligand contains 5or fewer amino acid differences, 10 or fewer amino acid differences, 25or fewer amino acid differences, 50 or fewer amino acid differences, or100 or fewer amino acid differences with respect to its naturallyoccurring counterpart. In certain embodiments of the invention thenumber of amino acid differences between a naturally occurringpolypeptide ligand and a fragment or variant thereof for use in theinvention is 5% or less, 10% or less, or 25% or less of the total numberof amino acids in the naturally occurring polypeptide.

In certain embodiments of the invention a fragment or variant of anaturally occurring polypeptide ligand is at least 70% identical, atleast 80% identical, at least 90% identical, at least 95% identical,over an amino acid portion that constitutes at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, or at least 90%, or 100% of the length of the naturallyoccurring counterpart. For example, variant that exhibits at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, or greatersequence identity, over the relevant portion of the sequence could beused, wherein % identity is determined as described above. The aminoacid portion is preferably at least 20 amino acids in length, morepreferably at least 50 amino acids in length. Alternately, a fragment orvariant can display significant or, preferably, substantial homology toa naturally occurring counterpart. Generally a fragment or variant of anaturally occurring polypeptide ligand possesses sufficient structuralsimilarity to its naturally occurring counterpart that it is recognizedby an antibody (e.g., a polyclonal or monoclonal antibody) thatrecognizes the naturally occurring counterpart. Peptide ligands can beidentified using phage display (Arap W, et al, Nature Medicine8(2):121-7, 2002); Zurita A J, et al., J Control Release, 91(1-2):183-6,2003; Pasqualini, R. & Ruoslahti, E. Nature 380, 364-366, 1996;Pasqualini, R., et al., Trends Mol. Med 8, 563-571, 2002).

In certain embodiments of the invention the ligand is an aptamer thatbinds to a cell type specific marker. In general, an aptamer is anoligonucleotide (e.g., DNA or RNA or) that binds to a particularprotein. Aptamers are typically derived from an in vitro evolutionprocess called SELEX, and methods for obtaining aptamers specific for aprotein of interest are known in the art. See, e.g., Brody E N, Gold L.J Biotechnol. 2000 March; 74(1):5-13.

Small molecules can also be used as ligands. Methods for identifyingsuch ligands are known in the art. For example in vitro screening ofsmall molecule libraries, including combinatorial libraries, andcomputer-based screening, e.g., to identify small organic compounds thatbind to concave surfaces (pockets) of proteins, can identify smallmolecule ligands for numerous proteins of interest (Huang, Z., Pharm. &Ther. 86: 201-215, 2000).

In certain embodiments of the invention binding moieties are notproteins or molecules that are typically used as carriers and conjugatedto antigens for the purpose of raising antibodies. Examples are carrierproteins or molecules such as bovine serum albumin, keyhole limpethemocyanin, bovine gamma globulin, and diphtheria toxin. In certainembodiments of the invention the cell binding moiety is not an Fcportion of an immunoglobulin molecule.

Methods for covalently or noncovalently linking a VCCP fragment orvariant to a binding moiety are known in the art and are described inU.S. Ser. No. 10/923,940. General methods for conjugation andcross-linking are described in “Cross-Linking”, Pierce ChemicalTechnical Library, available at the Web site having URL www. followedimmediately by piercenet.com and originally published in the 1994-95Pierce Catalog and references cited therein, in Wong S S, Chemistry ofProtein Conjugation and Crosslinking, CRC Press Publishers, Boca Raton,1991; and G. T. Hermanson, Bioconjugate Techniques, Academic Press,Inc., 1995. See also, Allen, T. M., Nature Reviews Cancer, 2, 750-763,2002, which describes methods of making targeted therapeutic agents. Forexample, according to certain embodiments of the invention abifunctional crosslinking reagent is used to couple a VCCP with anantibody or ligand. In general, bifunctional crosslinking reagentscontain two reactive groups, thereby providing a means of covalentlylinking two target groups. The reactive groups in a chemicalcrosslinking reagent typically belong to various classes includingsuccinimidyl esters, maleimides, pyridyldisulfides, and iodoacetamides.Bifunctional chelating agents may also be used.

Alternately, the VCCP or the VCCP fragment or variant and the moiety canbe produced as a fusion protein. Thus the invention provides a fusionprotein comprising: (i) a first domain comprising a VCCP or complementinhibiting VCCP fragment or variant; and (ii) a second domain comprisinga binding moiety that binds to a cellular marker or noncellularmolecular entity present in the eye of a subject suffering from or atrisk of a macular degeneration related condition or CNV. The firstdomain may be at the N or C terminus of the fusion protein. The fusionprotein may contain one or more additional domains at either the N or Cterminus or between the first and second domains.

Targeted VCCPs can be used for treatment of a number of conditions otherthan macular degeneration related conditions, diabetic retinopathy, orCNV. For such purposes the binding moiety need not bind to a site in theeye. In general, the binding moiety is selected to target the complementinhibiting protein to any site in the body at which complementinhibition is desired. For example, the compounds can be used to treatatherosclerosis, Alzheimer's disease, CNS injury (including spinal cordinjury), transplant rejection, or any other disease in which complementactivation plays a role (e.g., certain forms of glomerulonephritis,certain inflammatory conditions), etc. They can be used to preventcomplement activation during cardiac bypass surgery orischemia/reperfusion in myocardial infarction or stroke. Atheroscleroticplaques, organ transplants (e.g., xenotransplants, allotransplants,etc.) may be targeted. The targeted compositions can also be used invitro, e.g., to treat platelets (which are considered cells for purposesof the invention) or other blood preparations in order to inhibitcomplement, or to treat organs prior to transplantation. Appropriatebinding moieties, e.g., cell binding moieties or moieties that bind to acomponent in an atherosclerotic plaque, an Alzheimer's disease plaque(e.g., β-amyloid), etc. are used to target the VCCP to the plaque. A Gal(1,3-Gal) epitope on the surface of a transplanted organ can betargeted.

Modifications

VCCPs, VCIPs, VCCP or VCIP fragments or variants, and VCCPs, VCIPs, orVCCP or VCIP fragments or variants linked to a binding moiety can bemodified by addition of a molecule such as polyethylene glycol (PEG) orsimilar molecules to stabilize the compound, reduce its immunogenicity,increase its lifetime in the body, and/or increase its resistance todegradation. Methods for pegylation are well known in the art (Veronese,F. M. & Harris, Adv. Drug Deliv. Rev. 54, 453-456, 2002; Davis, F. F.,Adv. Drug Deliv. Rev. 54, 457-458 (2002; Hinds, K. D. & Kim, S. W. Adv.Drug Deliv. Rev. 54, 505-530 (2002; Roberts, M. J., Bentley, M. D. &Harris, J. M. Adv. Drug Deliv. Rev. 54, 459-476 (2002; Wang, Y. S. etal. Adv. Drug Deliv. Rev. 54, 547-570, 2002). A wide variety of polymerssuch as PEGs and modified PEGs, including derivatized PEGs to whichpolypeptides can conveniently be attached are described in NektarAdvanced Pegylation 2005-2006 Product Catalog, Nektar Therapeutics, SanCarlos, Calif., which also provides details of appropriate conjugationprocedures. Thus in some embodiments a VCCP, VCIP, or fragment orvariant of either is modified with one or more polypeptide ornon-polypeptide components, e.g., the VCCP, VCIP, or fragment or variantof either is pegylated or conjugated to another moiety. VCCPs, VCCPfragments and variants, and VCCPs or VCCP fragments or variants can beprovided as multimers or as part of a supramolecular complex.

Pharmaceutical Compositions and Delivery Vehicles and Methods

Suitable preparations, e.g., substantially pure preparations of a VCCP,VCIP, or a fragment or variant of either may be combined withpharmaceutically acceptable carriers, diluents, solvents, etc., toproduce an appropriate pharmaceutical composition. The inventiontherefore provides a variety of pharmaceutically acceptable compositionsfor administration to a subject comprising (i) a VCCP; and (ii) apharmaceutically acceptable carrier, adjuvant, or vehicle. The inventiontherefore provides a variety of pharmaceutically acceptable compositionsfor administration to a subject comprising (i) a complement inhibitingVCCP fragment or variant; and (ii) a pharmaceutically acceptablecarrier, adjuvant, or vehicle. The invention further provides apharmaceutically acceptable composition comprising (i) a VCCP linked toa moiety that binds to a component present on or at the surface of acell or noncellular molecular entity; and (ii) a pharmaceuticallyacceptable carrier, adjuvant, or vehicle. The moiety may be an antibodyor ligand. The component may be a marker such as a cell type specificmarker for RPE or endothelial cells, a drusen constituent, etc. Similarcompositions comprising a VCIP or a complement inhibiting fragment orvariant thereof are also provided.

In certain embodiments of the invention the pharmaceutical compositiondetectably inhibits neovascularization in an eye, followingadministration to a subject. In other words, administration of thecompound measurably reduces neovascularization relative to the expectedlevel in the absence of the composition. It is to be understood that thepharmaceutical compositions of the invention, when administered to asubject, are preferably administered for a time and in an amountsufficient to treat or prevent the disease or condition for whosetreatment or prevention they are administered.

Further provided are pharmaceutically acceptable compositions comprisinga pharmaceutically acceptable derivative (e.g., a prodrug) of any of thecompounds of the invention, by which is meant any non-toxic salt, ester,salt of an ester or other derivative of a compound of this inventionthat, upon administration to a recipient, is capable of providing,either directly or indirectly, a compound of this invention or aninhibitorily active metabolite or residue thereof. As used herein, theterm “inhibitorily active metabolite or residue thereof” means that ametabolite or residue thereof is also able to detectably inhibitcomplement, e.g., inhibit complement activation.

In various embodiments of the invention an effective amount of thepharmaceutical composition is administered to a subject by any suitableroute of administration including, but not limited to, intravenous,intramuscular, by inhalation, by catheter, intraocularly, orally,rectally, intradermally, by application to the skin, etc.

Inventive compositions may be formulated for delivery by any availableroute including, but not limited to parenteral, oral, by inhalation tothe lungs, nasal, bronchial, ophthalmic, transdermal (topical),transmucosal, rectal, and vaginal routes. The term “parenteral” as usedherein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered either locallyto the eye or intravenously.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat. Solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration maybe included. Supplementary active compounds, e.g., compoundsindependently active against the disease or clinical condition to betreated, or compounds that enhance activity of a compound, can also beincorporated into the compositions.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Salts derived from appropriate bases include alkali metal (e.g., sodiumand potassium), alkaline earth metal (e.g., magnesium), ammonium andN+(C1-4 alkyl)4 salts. This invention also envisions the quaternizationof any basic nitrogen-containing groups of the compounds disclosedherein. Water or oil-soluble or dispersible products may be obtained bysuch quaternization.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Solutions or suspensions used forparenteral (e.g., intravenous), intramuscular, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. For intravenous administration,suitable carriers include physiological saline, bacteriostatic water,Cremophor EL™ (BASF, Parsippany, N.J.), phosphate buffered saline (PBS),or Ringer's solution.

Sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or di-glycerides. Fatty acids, such as oleicacid and its glyceride derivatives are useful in the preparation ofinjectables, as are natural pharmaceutically-acceptable oils, such asolive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain a long-chain alcoholdiluent or dispersant, such as carboxymethyl cellulose or similardispersing agents that are commonly used in the formulation ofpharmaceutically acceptable dosage forms including emulsions andsuspensions. Other commonly used surfactants, such as Tweens, Spans andother emulsifying agents or bioavailability enhancers which are commonlyused in the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

In all cases, the composition should be sterile, if possible, and shouldbe fluid to the extent that easy syringability exists.

Preferred pharmaceutical formulations are stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. In general, therelevant carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin. Prolongedabsorption of oral compositions can be achieved by various meansincluding encapsulation.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Preferably solutions for injection are free ofendotoxin. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. Formulations fororal delivery may advantageously incorporate agents to improve stabilitywithin the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, the inventive compositions arepreferably delivered in the form of an aerosol spray from a pressuredcontainer or dispenser which contains a suitable propellant, e.g., a gassuch as carbon dioxide, or a nebulizer. Liquid or dry aerosol (e.g., drypowders, large porous particles, etc.) can be used. The presentinvention also contemplates delivery of compositions using a nasalspray.

For topical applications, the pharmaceutically acceptable compositionsmay be formulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutically acceptable compositions canbe formulated in a suitable lotion or cream containing the activecomponents suspended or dissolved in one or more pharmaceuticallyacceptable carriers. Suitable carriers include, but are not limited to,mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water.

For local delivery to the eye, the pharmaceutically acceptablecompositions may be formulated as micronized suspensions in isotonic, pHadjusted sterile saline, or, preferably, as solutions in isotonic, pHadjusted sterile saline, either with or without a preservative such asbenzylalkonium chloride. Alternatively, for ophthalmic uses, thepharmaceutically acceptable compositions may be formulated in anointment such as petrolatum.

Preferred methods of local administration include, e.g., choroidalinjection, transscleral injection or placing a scleral patch, selectivearterial catheterization, intraocular administration includingtransretinal, subconjunctival bulbar, intravitreous injection,suprachoroidal injection, subtenon injection, scleral pocket and scleralcutdown injection, by osmotic pump, etc. The agent can also bealternatively administered intravascularly, such as intravenously (IV)or intraarterially. In choroidal injection and scleral patching, theclinician uses a local approach to the eye after initiation ofappropriate anesthesia, including painkillers and ophthalmoplegics. Aneedle containing the therapeutic compound is directed into thesubject's choroid or sclera and inserted under sterile conditions. Whenthe needle is properly positioned the compound is injected into eitheror both of the choroid or sclera. When using either of these methods,the clinician can choose a sustained release or longer actingformulation. Thus, the procedure can be repeated only every severalmonths or several years, depending on the subject's tolerance of thetreatment and response.

Intraocular administration of drugs intended for treatment of maculardegeneration and other intraocular conditions is well known in the art.See, e.g., U.S. Pat. Nos. 5,632,984 and 5,770,589. U.S. Pat. No.6,378,526 provides methods for intrascleral injection of a therapeuticor diagnostic material at a location overlying the retina, which providea minimally invasive technique for delivering the agent to the posteriorsegment of the eye.

In certain embodiments of the invention a composition is delivered tothe vicinity of the eye, e.g., in close proximity to the posteriorsegment of the eye. The “vicinity of the eye” refers to locations withinthe orbit, which is the cavity within the skull in which the eye and itsappendages are situated. Typically the compositions would be deliveredclose to their intended target within the eye, e.g., close to (withinseveral millimeters of) the portion of the sclera that overlies theposterior segment of the eye, or immediately adjacent to the exteriorsurface of the sclera.

A number of polymeric delivery vehicles for providing controlled releasehave been used in an ocular context and can be used to administer thecompositions of the invention. Various polymers, e.g., biocompatiblepolymers, which may be biodegradable, can be used. For example, U.S.Pat. No. 6,692,759 describes methods for making an implantable devicefor providing controlled release of therapeutic agents in the eye. Otheruseful polymers and delivery systems for ocular administration of atherapeutic agent have been described. The active agent may be releasedas the polymer degrades. Polymers that have been used for drug deliveryinclude, but are not limited to, poly(lactic-co-glycolic acid),polyanhydrides, ethylene vinyl acetate, polyglycolic acid, chitosan,polyorthoesters, polyethers, polylactic acid, and poly (beta aminoesters). Peptides, proteins such as collagen and albumin, and dendrimers(e.g., PAMAM dendrimers) have also been used. Any of these can be usedin various embodiments of the invention.

Poly(ortho esters) have been introduced into the eye and demonstratedfavorable properties for sustained release ocular drug delivery(Einmahl, S., Invest. Ophthalmol. Vis. Sci., 43(5), 2002). Polylactideparticles have been used to target an agent to the retina and RPEfollowing intravitreous injection of a suspension of such particles(Bourges, J-L, et al, Invest. Ophthalmol. Vis. Sci., 44(8), 2003). Amacroscopic implantable device suitable for introduction into theposterior or anterior segment of the eye is referred to herein as anocular implant (Jaffe, G., Invest. Ophthalmol. Vis. Sci., 41(11), 2000;Jaffe, G., Ophthalmology). Such devices may be comprised of a pluralityof nanoparticles less than or microparticles impregnated with the agent.Methods for making microparticles and nanoparticles are known in theart. Generally, a microparticle will have a diameter of 500 microns orless, e.g., between 50 and 500 microns, between 20 and 50 microns,between 1 and 20 microns, between 1 and 10 microns, and a nanoparticlewill have a diameter of less than 1 micron. Preferably the device isimplanted into the space occupied by the vitreous humor. The ocularimplant may comprise a polymeric matrix. The invention also providesperiocular implants, which are macroscopic implantable device suitablefor introduction in the vicinity of the eye, e.g., in close proximity tothe eye. In certain embodiments the periocular implant is made ofsimilar materials to those described above.

As mentioned above, cells that express a VCCP, VCIP, or fragment orvariant of either, can be implanted into the eye. U.S. Pat. No.6,436,427 provides a method for delivering biologically active moleculesto the eye by implanting biocompatible capsules containing a cellularsource of the biologically active molecule.

The pharmaceutically acceptable compositions of this invention may alsobe administered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In addition to the agents described above, in certain embodiments of theinvention, the active compounds are prepared with carriers that willprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, polyethers, and polylactic acid. Methods forpreparation of such formulations will be apparent to those skilled inthe art. Certain of the materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensionscan also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811 and other referenceslisted herein. Liposomes, including targeted liposomes (e.g., antibodytargeted liposomes) and pegylated liposomes have been described (HansenC B, et al., Biochim Biophys Acta. 1239(2):133-44, 1995; Torchilin V P,et al., Biochim Biophys Acta, 1511(2):397-411, 2001; Ishida T, et al.,FEBS Lett. 460(1):129-33, 1999). One of ordinary skill in the art willappreciate that the materials and methods selected for preparation of acontrolled release formulation, implant, etc., should be such as toretain activity of the compound. For example, it may be desirable toavoid excessive heating of polypeptides, which could lead todenaturation and loss of activity.

The invention also encompasses gene therapy, in which a nucleic acidthat encodes a VCCP, VCIP, or fragment or variant of either in operableassociation with regulatory elements sufficient to direct expression ofthe fragment or variant is introduced into a subject. Nucleic acids canbe introduced into a subject by any of a number of methods. Forinstance, a pharmaceutical preparation of a nucleic acid therapeutic canbe introduced systemically, e.g., by intravenous injection. Expressionof the polypeptide in particular target cells may result fromspecificity of transfection provided by the vector, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the gene, or a combination thereof.Alternatively, initial delivery of the nucleic acid can be more limited.For example, the vector can be locally introduced into the eye using anyof the methods described above for ocular administration.

A pharmaceutical composition comprising a nucleic acid therapeutic ofthe invention can consist essentially of the nucleic acid or a genetherapy vector comprising in an acceptable diluent, or can comprise aslow release matrix in which the nucleic acid or gene therapy vector isencapsulated or embedded. The gene therapy vector can be a plasmid,virus, or other vector. Alternatively, the pharmaceutical compositioncan comprise one or more cells which produce a therapeutic nucleic acidor polypeptide such as a VCCP, VCIP, or fragment or variant of either.Preferably such cells secrete the fragment or variant thereof into theextracellular space or bloodstream.

Viral vectors that have been used for gene therapy protocols include,but are not limited to, retroviruses, lentiviruses, other RNA virusessuch as poliovirus or Sindbis virus, adenovirus, adeno-associated virus,herpes viruses, SV 40, vaccinia and other DNA viruses.Replication-defective murine retroviral or lentiviral vectors are widelyutilized gene transfer vectors. Chemical methods of gene therapy involvecarrier-mediated gene transfer through the use of fusogenic lipidvesicles such as liposomes or other vesicles for membrane fusion. Acarrier harboring a nucleic acid of interest can be convenientlyintroduced into the eye or into body fluids or the bloodstream. Thecarrier can be site specifically directed to the target organ or tissuein the body. Cell or organ-specific DNA-carrying liposomes, for example,can be developed and the foreign nucleic acid carried by the liposomeabsorbed by those specific cells. Carrier mediated gene transfer mayalso involve the use of lipid-based compounds which are not liposomes.For example, lipofectins and cytofectins are lipid-based compoundscontaining positive ions that bind to negatively charged nucleic acidsand form a complex that can ferry the nucleic acid across a cellmembrane. Cationic polymers are known to spontaneously bind to andcondense nucleic acids such as DNA into nanoparticles. For example,naturally occurring proteins, peptides, or derivatives thereof have beenused. Synthetic cationic polymers such as polyethylenimine (PEI),polylysine (PLL) etc., are also known to condense DNA and are usefuldelivery vehicles. Dendrimers can also be used.

Many of the useful polymers contain both chargeable amino groups, toallow for ionic interaction with the negatively charged DNA phosphate,and a degradable region, such as a hydrolyzable ester linkage. Examplesof these include poly(alpha-(4-aminobutyl)-L-glycolic acid), networkpoly(amino ester), and poly(beta-amino esters). These complexationagents can protect DNA against degradation, e.g., by nucleases, serumcomponents, etc., and create a less negative surface charge, which mayfacilitate passage through hydrophobic membranes (e.g., cytoplasmic,lysosomal, endosomal, nuclear) of the cell. Certain complexation agentsfacilitate intracellular trafficking events such as endosomal escape,cytoplasmic transport, and nuclear entry, and can dissociate from thenucleic acid. It has been proposed that such agents may act as a “protonsponge” within the endosome.

It is typically advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

A therapeutically effective amount of a pharmaceutical compositiontypically ranges from about 0.001 to 100 mg/kg body weight, preferablyabout 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. Thepharmaceutical composition can be administered at various intervals andover different periods of time as required, e.g., multiple times perday, daily, every other day, once a week for between about 1 to 10weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or6 weeks, etc. The skilled artisan will appreciate that certain factorscan influence the dosage and timing required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Generally, treatment of a subjectwith an inventive composition can include a single treatment or, in manycases, can include a series of treatments.

Exemplary doses include milligram or microgram amounts of the inventivecompounds per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram.) For localadministration (e.g., intranasal), doses much smaller than these may beused. It is furthermore understood that appropriate doses depend uponthe potency of the agent, and may optionally be tailored to theparticular recipient, for example, through administration of increasingdoses until a preselected desired response is achieved. It is understoodthat the specific dose level for any particular subject may depend upona variety of factors including the activity of the specific compoundemployed, the age, body weight, general health, gender, and diet of thesubject, the time of administration, the route of administration, therate of excretion, any drug combination, and the degree of expression oractivity to be modulated.

The invention further provides pharmaceutical compositions comprisingtwo or more molecular species of the invention, each comprising a moietythat binds to a cellular marker on noncellular molecular entity, whereinthe binding moieties in each molecular species bind to a differentcellular marker. The invention further provides pharmaceuticalcompositions comprising one or more molecular species of the inventionand an additional active agent. The additional active agent may be anagent that is effective for treatment of a macular degeneration relatedcondition, diabetic retinopathy, or CNV. In certain embodiments of theinvention the additional active agent is selected from the groupconsisting of: angiogenesis inhibitors, antiinflammatory agents,antiangiogenic steroids, and growth factors. Angiogenesis inhibitors arediscussed further below. The additional active agent can be anantibiotic or an antiinflammatory agent not necessarily effectivespecifically for treatment of a macular degeneration related condition,diabetic retinopathy, or CNV.

Angiogenesis Inhibitors

Certain embodiments of the present invention make use of one or moreangiogenesis inhibitors. Angiogenesis inhibitors can be divided intoseveral groups based on their primary mechanism of action. One groupincludes cytotoxic agents that damage or kill target cells (e.g.,endothelial cells) or that trigger an immune-mediated response thatresults in damage to or killing of target cells. A second group includesagents that do not substantially damage or kill endothelial cells butinstead inhibit their proliferation, migration, capillary tubeformation, or other processes associated with angiogenesis. Angiogenesisinhibitors falling into either or both of these groups can be used.

Angiogenesis inhibitors include, but are not limited to, Macugen® oranother VEGF nucleic acid ligand; Lucentis®, Avastin®, or anotheranti-VEGF antibody; combretastatin or a derivative or prodrug thereofsuch as Combretastatin A4 Prodrug (CA4P); VEGF-Trap; EVIZON™ (squalaminelactate); AG-013958 (Pfizer, Inc.); JSM6427 (Jerini AG); a shortinterfering RNA (siRNA) that inhibits expression of one or more VEGFisoforms (e.g., VEGF₁₆₅); and an siRNA that inhibits expression of aVEGF receptor (e.g., VEGFR1). Other angiogenesis inhibitors includevarious endogenous or synthetic peptides such as angiostatin, arresten,canstatin, combstatin, endostatin, thrombospondin, and tumstatin. Otherantiangiogenic molecules include thalidomide and its antiangiogenicderivatives such as iMiDs (Bamias A, Dimopoulos M A. Eur J Intern Med.14(8):459-469, 2003; Bartlett J B, Dredge K, Dalgleish A G. Nat RevCancer. 4(4):314-22, 2004). β2-glycoprotein 1 (β2-GP1) is anangiogenesis inhibitor of particular interest in the present invention.

Macugen (Pfizer, Eyetech) is a VEGF nucleic acid ligand (also referredto as an aptamer) that binds to and inhibits VEGF₁₆₅ (U.S. Pat. No.6,051,698). Lucentis (Genentech) is a humanized antibody fragment thatbinds and inhibits Vascular Endothelial Growth Factor A (VEGF-A).(Gaudreault, J., et al., Invest Ophthalmol. Vis. Sci. 46, 726-733 (2005)and references therein. Avastin (Genentech) is a full length humanizedantibody that also binds to VEGF. Cand5 (Acuity Pharmaceuticals,Philadelphia, Pa.) is a short interfering RNA (siRNA) designed toinhibit expression of VEGF. sima-027 (Sima Therapeutics; Boulder Colo.)is a chemically modified siRNA designed to inhibit expression of theVEGF receptor known as VEGFR1.

β2-GP1, also known as apolipoprotein H, (apoH) (GenBank entry for thecomplete human β2 glycoprotein 1 is NP_(—)000033), is an abundant plasmaglycoprotein that circulates either as a free protein or associated withlipoproteins. (Polz et al., FEBS Letters 102:183-186, 1979; Wurm, H.,Int. J. Biochem. 16:511-515, 1984). It is a ˜54-kDa single-chainglycoprotein consisting of 326 amino acids. β2-GP1 and methods for itsproduction are further described in U.S. Pub. No. 20030219406. As usedherein, the term β2-GP1 is used to refer to both the intact form ofβ2-GP1 and the nicked form of β2-GP1. Nicked β2-GP1 is a β2-GP1polypeptide that is cleaved at Lys 317/Thr 318. In a preferredembodiment, the portions of nicked β2-GP1 remain linked by disulfidebond(s). As used herein, the term β2-GP1 polypeptide includes β2-GP1having a sequence identical to the native human form (SEQ ID NO: 13) andvariants and fragments thereof. The amino acid sequence of β2-GP 1varies between species, and such variations fall within the scope of theterm “β2-GP1”. In certain embodiments of the invention a variant orfragment has significant or substantial sequence homology to nativehuman β2-GP1. In specific embodiments a β2-GP1 polypeptide is at least80% identical to SEQ ID NO: 13, or at least 90% identical to SEQ ID NO:13 over at least 80%, at least 90%, or approximately 100%, e.g., 100%,of SEQ ID NO: 13. In some embodiments a fragment lacks one or more ofdomains I-V of SEQ ID NO: 13.

Compositions Comprising a VCCP or VCIP and a Gel-Forming Material

The invention provides a variety of compositions comprising a solublegel-forming and a therapeutic agent, wherein said therapeutic agent iseffective for treating an eye disorder characterized by maculardegeneration, CNV, RNV, or any combination of these. In variousembodiments of the invention the therapeutic agent is a VCCP or VCIP, ora complement inhibiting fragment or variant of either. The compositionmay comprise one or more additional therapeutic agents effective fortreating the eye disorder. Suitable agents are described elsewhereherein.

The invention encompasses the recognition that compositions comprising asoluble gel-forming material are of particular use for the delivery ofbiological macromolecules such as polypeptides, polynucleotides, orcarbohydrates to the posterior segment of the eye. Certain embodimentsof the invention therefore provide a uniquely favorable system fordelivery of macromolecules such as polypeptides or polynucleotides tothe posterior segment of the eye for treatment of eye disorders. Thegel-forming material is initially at least partially soluble but iscapable of forming a gel under appropriate conditions. The system isdesigned to localize biological macromolecules in sufficientconcentration to provide sustained delivery while at the same timeallowing the macromolecule to be released in sufficient amounts so thatit can diffuse to a site of action in the posterior segment of the eye,e.g., the retina, RPE, subretinal space, Bruch's membrane, and/orchoriocapillaris. In addition, the gel may protect the biologicalmacromolecule from degradation, e.g., by endogenous proteases ornucleases.

A variety of biological macromolecules useful for the treatment of eyedisorders characterized by macular degeneration, CNV, RNV, or anycombination of the foregoing, can be delivered using the gel-formingcompositions of the invention. Any of the agents mentioned herein, e.g.,angiogenesis inhibitors such as Macugen, Lucentis, β2-GP1, etc., can bedelivered either singly or in combination with one or more other agents.The compositions can also be used to deliver agents that are notbiological macromolecules. The invention therefore provides acomposition comprising: (i) a therapeutic agent effective for thetreatment of an eye disorder characterized by macular degeneration, CNV,RNV, or any combination of the foregoing; and (ii) a soluble gel-formingmaterial. In certain embodiments of the invention the agent is acomplement inhibitor, e.g., a VCCP or VCIP. The complement inhibitormay, but need not be, a polypeptide or peptide.

In accordance with certain embodiments of the invention, a solutioncontaining the gel-forming material and a therapeutic agent is preparedby combining the gel-forming material and therapeutic agent in solutionusing any suitable method, e.g., by adding the therapeutic agent to asolution containing the gel-forming material. In certain embodiments thecomposition forms a gel following introduction into the body, e.g., uponcontact with a physiological fluid. The composition can also form a gelupon contact with a fluid such as phosphate buffered saline, or otherfluid containing appropriate ions. Thus the composition can be injectedat an appropriate location, e.g., in close proximity to the posteriorsegment of the eye, where it forms a gel. Alternately, a preshaped gelimplant can be made, e.g., by introducing the solution into a mold orcavity of the desired shape and allowing gel formation to occur in thepresence of a suitable concentration of a salt. The salt can be addedeither prior to or following the introduction of the solution into themold or cavity. The mold or cavity can be, e.g., any structure thatcontains a hollow space or concave depression into which a solution canbe introduced. In another embodiment, a film or membrane is formed fromthe gel-forming solution containing a therapeutic agent.

Release of the agent from the gel can occur by any mechanism, e.g., bydiffusion of the agent out of the gel, as a result of breakdown of thegel, or both. In certain embodiments of the invention the gel-formingmaterial also comprises at least some solid material in addition tosoluble material.

A variety of different gel-forming materials can be used in the presentinvention. Preferably the gel is a hydrogel, by which is meant a gelthat contains a substantial amount of water. Preferably the material andthe gel that it forms are biocompatible. Preferably the material and thegel that it forms are biodegradable.

In certain embodiments of the invention soluble collagen is used as thegel-forming material. The invention encompasses the recognition thatgel-forming compositions comprising a soluble collagen are particularlyadvantageous for the delivery of biological macromolecules such aspolypeptides, polynucleotides, or carbohydrates to the posterior segmentof the eye. The collagen is initially soluble, e.g., in an aqueousmedium, and forms a solution that has a low viscosity but is capable ofrapid formation of a gel under appropriate conditions, e.g., conditionsencountered upon administration to a mammalian subject. In accordancewith certain embodiments of the invention, a solution containing thesoluble collagen and a therapeutic agent is prepared by combining thesoluble collagen and therapeutic agent in solution using any suitablemethod, e.g., by adding the therapeutic agent to a solution containingsoluble collagen. The composition is delivered locally to an appropriatelocation in or near the eye of a mammalian subject, typically to an areaoutside of and in close proximity to the posterior segment of the eye.The solution rapidly forms a gel at or close to of the site ofadministration. The therapeutic agent is entrapped within the gel. Thetherapeutic agent diffuses out of the gel or is released as the geldegrades over time, thereby providing a continuous supply of the agentto tissues and structures that are either in direct physical contactwith the gel or located nearby. In certain embodiments the solution isadministered behind the sclera of the eye, as discussed further below.Delivery can be accomplished by injection (e.g., using a 30 gauge needleor the like), by catheter, etc., as further described below.

A variety of different collagen preparations can be used in the presentinvention provided that the collagen is initially soluble and is capableof rapidly forming a gel under appropriate conditions. Suitable collagenpreparations, and methods for their manufacture, are described, e.g., inU.S. Pat. Nos. 5,492,135; 5,861,486; 6,197,934; 6,204,365; and WO00/47130, but the invention is not limited to such preparations ormethods. These collagens are prepared in soluble form and rapidly form agel upon exposure to physiological fluids or other fluids havingsuitable concentration of ions. In accordance with the presentinvention, injecting or otherwise introducing the collagen solution tothe eye or near the eye results in gel formation, presumably induced bycontact with physiological fluids. However it is noted that theinvention is in no way limited by the mechanism by which gel formationoccurs. In addition, as noted above, the gel can be formed in vitro andthen implanted at an appropriate location, e.g., in close proximity tothe posterior segment of the eye.

One suitable method of preparing a soluble collagen solution involvesextracting collagen from a natural source, acid solubilizing thecollagen, and dialyzing the solubilized collagen against a solutioncontaining a chelating agent, e.g., a metal chelating agent such asethylenediamine tetraacetic acid, disodium salt dihydrate (EDTA), whileraising the pH. One or more dialysis steps against a solution such asdeionized water lacking the chelating agent may also be performed.Unlike standard collagen solutions that undergo spontaneousfibrillogenesis at neutral pH and room temperature, collagen solutionsfor use in the present invention remain in solution during storage forextended periods of time and rapidly undergo gel formation when exposedto physiological fluids. While not wishing to be bound by any theory,the chelating agent may alter the concentration of one or more cationsand thereby prevent fibrillogenesis that would otherwise occur as the pHis raised. The chelating agent may have other desirable effects on thecollagen solution, and in certain embodiments of the invention thecollagen solution comprises a chelating agent, e.g., EDTA. The chelatingagent may remain in the collagen solution following dialysis or may beadded to the collagen solution. The concentration of the chelating agentmay range, for example, between about 0.02 M and about 0.05 M, e.g.,between about 0.025 M and about 0.035 M. Other chelating agents may alsobe used including, but not limited to, those described in U.S. Pat. No.5,861,486.

In certain embodiments the collagen solution has a concentration ofsoluble collagen ranging between 1 mg/ml and 100 mg/ml, e.g., between 10mg/ml and 70 mg/ml, between 20 mg/ml and 50 mg/ml, e.g., 30 mg/ml, etc.In certain embodiments of the invention the pH of the collagen solutionis between 6.0 and 8.0, e.g., between 6.5 and 7.5, e.g., 7.0.

In certain embodiments of the invention the collagen composition furthercomprises a fibrillar component comprising collagen solids, e.g.,fibrillar collagen solids. One aspect of the invention is the selectionof suitable concentrations of soluble collagen and collagen solids thatresult in a gel that retains the agent within the gel so as to providesustained delivery for a desired period of time while also permittingrelease of the agent from the gel in sufficient concentration to beeffective at its site of action in the posterior segment of the eye. Forexample, certain collagen compositions contain between 0.5 mg/ml and 30mg/ml fibrillar collagen solids, or between 1 mg/ml and 20 mg/mlfibrillar collagen solids, e.g., 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6mg/ml, 8 mg/ml, 10 mg/ml, etc. In terms of percent fibrillar collagensolids on a weight/volume basis, certain collagen compositions containbetween 0.05 and 3% fibrillar collagen solids or between 0.1 and 2%fibrillar collagen solids, e.g., 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%,1.2%, etc. Any suitable fibrillar component can be used in the collagencompositions of the invention. Fibrillar collagen solids can be preparedusing a variety of methods. For example, fibrillar collagen may bereconstituted collagen prepared from animal sources such as bovine hide(Frontiers in Matrix Biology, Vol. 10, pp. 1-58, in Methods ofConnective Tissue Research, Eds. Robert, Moczar, and Moczar, S. Karger,Basel, 1985). Fibrillar collagen may be prepared from human or animalsources as described in U.S. Pat. Nos. 4,969,912 and 5,322,802. Thefibrillar collagen solids are suspended in solution at a concentrationtypically ranging from about 10-100 mg/ml. The collagen suspensioncontaining fibrillar collagen solids is combined with, e.g., added to, asoluble collagen composition either prior to or following addition ofthe therapeutic agent to a solution comprising soluble collagen.

Without wishing to be bound by any theory, the presence of fibrillarcollagen solids may have any of a variety of advantageous effects. Byway of non-limiting example, the fibrillar collagen solids may increasethe in vivo stability of the collagen gel, e.g., they may decrease therate of breakdown of the gel. The fibrillar collagen solids may increasethe stability of a therapeutic agent contained in the gel and/ordecrease or modulate the rate at which the agent is released from thegel by diffusion and/or breakdown of the gel.

In some embodiments of the invention the soluble collagen preparationcomprises a chemical cross-linking agent. The agent may crosslinkcollagen molecules and/or fibrils to one another and/or may crosslink atherapeutic agent such as a VCCP or VCIP to a collagen molecule orfibril. Typical cross-linking agents crosslink collagen amine groups toone another or to amine, carboxyl, phenol, sulfonyl, or carbohydrategroups of therapeutic agents. Suitable cross-linking agents include, butare not limited to, those described in WO 00/47130. Without wishing tobe bound by any theory, cross-linking may stabilize the collagen gel(e.g., decrease its rate of breakdown) and/or decrease the rate ofrelease of the therapeutic agent from the gel.

The collagen preparations preferably form a gel within 5 minutes (300seconds) following contact with physiological fluids. More preferablythe collagen preparations form a gel within 90 seconds, 2 minutes (120seconds) or within 3 minutes (180 seconds) following contact withphysiological fluids. Preparations that form a gel within shorter timeperiods, e.g., within 5-90 seconds, or longer time periods, e.g., 3-5minutes, can also be used.

Any of collagen types I-XXVIII, or mixtures thereof, can be used in thepresent invention. The collagen can be purified from natural sources(e.g., human tissue or animal tissue such as bovine, rabbit, etc.) asdescribed in the above-referenced patents and publications.Alternatively, the collagen can be manufactured using recombinant DNAtechniques, in which case the sequence can be of human or animal origin.See, e.g., U.S. Pat. Nos. 5,593,854 and 5,667,839. Methods for theproduction of proteins, e.g., a polypeptide of interest such as acollagen chain, using recombinant DNA technology are well known in theart. Suitable methods include those described above. The term “collagen”includes collagen fragments. Thus in certain embodiments the solublecollagen comprises or consists of a collagen fragment or combination offragments. In certain embodiments a complete collagen polypeptide chainis used.

A variety of modified or derivatized collagens are also of use invarious embodiments of the invention. See, e.g., U.S. Pat. No.5,201,764. For example, collagen can be acylated with one or moreacylating agents such as glutaric anhydride, succinic anhydride, andmaleic anhydride and at least one other acylating agent selected fromthe group consisting of methacrylic anhydride, beta-styrene sulfonylchloride, ethylene-maleic anhydride copolymer, styrene-maleic anhydridecopolymer or poly(vinyl) sulfonic acid.

Other gel-forming materials of use in the invention include, but are notlimited to, hyaluronic acid and modified forms thereof, polysaccharidessuch as alginate and modified forms thereof, self-assembling peptides,etc. See, e.g., U.S. Pat. No. 6,129,761 for further description ofalginate and modified forms thereof, hyaluronic acid and modified formsthereof, and additional examples of soluble gel-forming materials thatare of use in various embodiments of the present invention. As describedtherein, other polymeric hydrogel precursors include polyethyleneoxide-polypropylene glycol block copolymers such as Pluronics™ orTetronics™ which are crosslinked by hydrogen bonding and/or by atemperature change, as described in Steinleitner et al., Obstetrics &Gynecology, 77:48-52 (1991); and Steinleitner et al., Fertility andSterility, 57:305-308 (1992). Other materials which may be utilizedinclude proteins such as fibrin or gelatin. Polymer mixtures also may beutilized. For example, a mixture of polyethylene oxide and polyacrylicacid which gels by hydrogen bonding upon mixing may be utilized.

Typically a gel-forming material of use in the invention is capable ofbeing at least partly dissolved, or in certain embodiments of theinvention substantially or fully dissolved, e.g., in an aqueous medium.For example, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or more, by weight, of the gel-forming materialpresent in a gel-forming composition may be dissolved. In certainembodiments essentially 100% of the material is dissolved.

Covalently crosslinkable hydrogel precursors also are useful. Forexample, a water soluble polyamine, such as chitosan, can becross-linked with a water soluble diisothiocyanate, such as polyethyleneglycol diisothiocyanate. The isothiocyanates will react with the aminesto form a chemically crosslinked gel. Aldehyde reactions with amines,e.g., with polyethylene glycol dialdehyde also may be utilized. Ahydroxylated water soluble polymer also may be utilized.

Alternatively, polymers may be utilized which include substituents whichare crosslinked by a radical reaction upon contact with a radicalinitiator. For example, polymers including ethylenically unsaturatedgroups which can be photochemically crosslinked may be utilized, asdisclosed in WO 93/17669, the disclosure of which is incorporated hereinby reference. In this embodiment, water soluble macromers that includeat least one water soluble region, a biodegradable region, and at leasttwo free radical-polymerizable regions, are provided. The macromers arepolymerized by exposure of the polymerizable regions to free radicalsgenerated, for example, by photosensitive chemicals and or light.Examples of these macromers are PEG-oligolactyl-acrylates, wherein theacrylate groups are polymerized using radical initiating systems, suchas an eosin dye, or by brief exposure to ultraviolet or visible light.Additionally, water soluble polymers which include cinnamoyl groupswhich may be photochemically crosslinked may be utilized, as disclosedin Matsuda et al., ASAID Trans., 38:154-157 (1992).

In general, the polymers are at least partially soluble in aqueoussolutions, such as water, buffered salt solutions, or aqueous alcoholsolutions. Methods for the synthesis of the other polymers describedabove are known to those skilled in the art. See, for example ConciseEncyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts,E. Goethals, editor (Pergamen Press, Elmsford, N.Y. 1980). Manypolymers, such as poly(acrylic acid), are commercially available.Naturally occurring and synthetic polymers may be modified usingchemical reactions available in the art and described, for example, inMarch, “Advanced Organic Chemistry,” 4th Edition, 1992,Wiley-Interscience Publication, New York.

Water soluble polymers with charged side groups may be crosslinked byreacting the polymer with an aqueous solution containing ions of theopposite charge, either cations if the polymer has acidic side groups oranions if the polymer has basic side groups. Examples of cations forcrosslinking of the polymers with acidic side groups to form a hydrogelare monovalent cations such as sodium, and multivalent cations such ascopper, calcium, aluminum, magnesium, strontium, barium, and tin, anddi-, tri- or tetra-functional organic cations such as alkylammoniumsalts. Aqueous solutions of the salts of these cations are added to thepolymers to form soft, highly swollen hydrogels and membranes. Thehigher the concentration of cation, or the higher the valence, thegreater the degree of cross-linking of the polymer. Additionally, thepolymers may be crosslinked enzymatically, e.g., fibrin with thrombin.In some embodiments a self-assembling peptide, such as those describedin U.S. Pat. No. 6,800,481 is used. These peptides self-assemble to forma hydrogel structure upon contact with monovalent cations, e.g., such asthose present in extracellular fluid.

In embodiments of the invention in which the gel is formed bycross-linking polymer chains to one another, the composition can includean appropriate cross-linking agent, which is selected according to theparticular polymer. Alternately, the cross-linking agent can beadministered after administration of the composition containing thegel-forming material, at substantially the same location. Any of thesegels can be formed in vitro, e.g., as described above for gels, andimplanted at an appropriate location in the eye or in the vicinity ofthe eye.

In certain embodiments of the invention the composition contains cellsthat produce and secrete a VCCP or VCIP or fragment or variant of eitherinstead of, or in addition to, containing the VCCP or VCIP or fragmentor variant itself. In these embodiments, the gel may be resistant todegradation, so that it traps the cells therein for a sustained periodof time.

Methods of Administration, Dose, and Dosing Regimens for a CompositionComprising a Gel-Forming Material

Any suitable method may be used to administer the gel-formingcompositions of the invention to a location in or near the posteriorsegment of the eye. As shown in FIGS. 1A and 1B, the eye can be dividedinto an anterior segment and a posterior segment. The sclera, which is athin, avascular layer of tissue, covers the outside of the eye aroundthe posterior segment and part of the anterior segment and is continuouswith the cornea, the transparent covering of the front of the eye. Thechoroid and retina underlie the sclera. The optic nerve transmits nerveimpulses from the retina along the visual pathways.

The composition may be administered by a periocular approach, which termis used to refer to any route of administration that locally delivers acomposition into the region outside the eye, i.e., exterior to thesclera. The composition is thus delivered to an area outside of and inclose proximity to the posterior segment of the eye. In certainembodiments a composition administered in close proximity to theposterior segment of the eye is administered such that at least one edgeor surface of the gel is within 10 mm of at least one point on theexterior surface of the portion of the sclera that covers the outside ofthe posterior segment of the eye. Preferably at least one edge orsurface of the gel is within 5 mm of at least one point on the exteriorsurface of the portion of the sclera that covers the outside of theposterior segment of the eye. In certain embodiments at least one edgeor surface of the gel is within 1-2 mm of at least one point on theexterior surface of the portion of the sclera that covers the outside ofthe posterior segment of the eye, or within 1 mm or less of at least onepoint on the exterior surface of the portion of the sclera that coversthe outside of the posterior segment of the eye.

Periocular administration may be accomplished using, e.g., retrobulbar,peribulbar, sub-Tenon, or subconjunctival injection, by subretinalinjection, by suprachoroidal injection, or by use of a catheter orcannula directed to any of the regions accessed by the afore-mentionedtechniques. Most commonly a syringe is used, but a pump or any othersource of pressure could also be used. In certain preferred embodimentsof the invention the composition is administered adjacent to the sclera,outside the eye, e.g., by retrobulbar, sub-Tenon, or subconjunctivalinjection. At least one surface of the gel may be in direct contact withthe sclera. Methods suitable for administration of local anesthesia forophthalmic surgery are of use to deliver a composition of the invention.See, e.g., Dutton, J J, et al., “Anesthesia for intraocular surgery”,Surv Ophthalmol. 46(2):172-84, 2001; Canavan, K. S., et al.,“Sub-Tenon's administration of local anaesthetic: a review of thetechnique”, British Journal of Anaesthesia, 90(6), 787-793, 2003. Seealso, Spaeth, supra, and Albert and Lucarelli, supra. Compositionsdelivered according to these standard techniques are considered to bedelivered in close proximity to the posterior segment of the eye. Thecomposition forms a gel which, in certain embodiments of the inventionat least partially overlies the macula. In certain embodiments of theinvention the composition is administered into the sclera itself, e.g.,by injection or using a catheter or cannula (see, e.g., U.S. Pat. No.6,378,526). The therapeutic agent is released from the composition anddiffuses from its site of release across the sclera and into the eye,where it reaches a site of activity at the retina. Alternately, a gelstructure formed in vitro can be implanted in or in the vicinity of theeye.

The amount and concentration of the therapeutic agent(s) in acomposition comprising a gel-forming material can vary depending on anumber of factors including, but not limited to, the identity of thetherapeutic agent(s), the condition being treated and its severity, thepresence or absence of solids and/or chemical cross-linking agents inthe composition, the total amount of composition administered (whichitself can vary based on various considerations such as the anatomy ofthe patient, etc.) For example, in the case of a composition comprisingsoluble collagen, the amount and concentration may vary depending uponthe amount of fibrillar collagen in the composition. It may be desirableto employ a concentration and/or total amount of therapeutic agent(s)that will maximize the total amount of agent delivered to the eye, whilekeeping the concentration actually released from the gel below thatwhich could cause unacceptable side effects. In certain embodiments ofthe invention the total amount and concentration of the agent(s) areselected to provide an effective concentration of the agent at theretina over a period of at least 4 weeks, e.g., 4-6 weeks, 6 weeks orgreater, 8 weeks or greater, etc.

The dosing interval (i.e., the time between individual administrationsof an inventive composition) and the dose of the therapeutic agentdelivered with each administration can vary. In certain embodiments thecomposition is delivered at times more than 6 weeks apart, e.g., 2, 3,4, 5, or 6 months apart, or any intervening number of weeks, e.g., 8,10, 12, 14, 16 weeks, etc. In other embodiments the composition isdelivered at even greater time intervals, e.g., at times 7, 8, 9, 10,11, or 12 months apart. In other embodiments the time interval is 6weeks or less. Of course the time interval can vary. For example, thetime intervals between doses can alternate between 6 weeks or less andmore than 6 weeks. In certain embodiments the average time intervalbetween administrations of an inventive composition is at least 6 weeks,e.g., 2, 3, 4, 5, or 6 months, or any intervening number of weeks, e.g.,8, 10, 12, 14, 16 weeks, etc. In certain embodiments of the inventionthe composition is administered multiple times at time intervals onaverage at least 6 weeks apart, at least 8 weeks apart, at least 10weeks apart, at least 12 weeks apart, etc. Typically the composition isadministered at least 2, 5, 10, 20, 50, or more times. The compositioncan and often will be administered indefinitely to a subject sufferingfrom or at risk of a macular degeneration related condition, CNV, RNV,etc.

The total amount of therapeutic agent and its concentration in the gelcan also vary. Exemplary, nonlimiting, doses are between approximately0.1 and 100 mg/dose for each eye to be treated, e.g., betweenapproximately 0.5 and 50 mg/dose, between 1 and 10 mg/dose, etc.Exemplary, nonlimiting concentrations of a therapeutic agent in acomposition of the invention are between approximately 0.1 and 100 mg ofthe therapeutic agent per milliliter of collagen solution, e.g., theconcentration may be between 1 and 50 mg/ml, between 1 and 10 mg/ml,etc.

In some embodiments a dose of a first therapeutic agent such as a VCCPor VCIP is administered intravitreally, and a composition of theinvention comprising a second therapeutic agent, which can be the sameas or different to the first therapeutic agent, is administered to thesubject using a periocular administration technique, with the twoadministrations occurring within a reasonably narrow period of time,e.g., within up to about 6 weeks of one another. The intravitrealadministration may provide an initial high concentration of thetherapeutic agent at the retina. The periocular administration thenprovides a sustained release of the therapeutic agent over time.

Testing Therapeutic Potential in Animal Models

A number of different animal models that attempt to replicate one ormore features of macular degeneration, diabetic retinopathy, and/orchoroidal neovascularization are known in the art. A compound of theinvention can be administered in various doses to mice, rats, dogs,primates, etc. that have either spontaneous or macular degenerationand/or choroidal neovascularization or in which macular degenerationand/or choroidal neovascularization have been induced by a treatment.The ability of the compound to prevent or treat one or more signs orsymptoms of macular degeneration (e.g. CNV, accumulation of lipofuscinin and/or drusen beneath the RPE, photoreceptor atrophy or hypertrophy,altered RPE pigmentation, photoreceptor loss, altered electroretinogram,etc.) is assessed. Visual examination, photography, histopathology,immunohistology, etc., can be used.

Useful models include animals (e.g., mice, Yucatan pigs, etc.) in whichchoroidal neovascularization is induced by laser treatment (Bora, P. S.,et al., Proc. Natl. Acad. Sci. 100(5): 2679-2684, 2003; Zacks, D N, etal., Invest Ophthalmol Vis Sci. 243(7):2384-91, 2002). Other modelsinclude animals that have been treated with a variety of agents such aslipid hydroperoxide (Tamai, K., et al., Exp Eye Res. 74(2):301-8, 2002),pellets comprising growth factors, etc. Animals genetically engineeredto overexpress or underexpress one or more genes are also useful. Forexample, transgenic mice (mcd/mcd mice) that express a mutated form ofcathepsin D that is enzymatically inactive display features associatedwith geographic atrophy (Rakoczy, P E, et al, Am. J. Path., 161(4),1515-1524, 2002). Adeno-associated virus (AAV) mediated expression ofvascular endothelial growth factor induces choroidal neovascularizationin rats (Wang, F., et al., Invest Ophthalmol Vis Sci. 44(2):781-90,2003). One animal model is a transgenic mouse deficient in eithermonocyte chemoattractant protein (Ccl-2) or its cognate chemokinereceptor (Ccr-2) (Ambati, J., et al., Nat Med. 9(11):1390-7, 2003; U.S.Ser. No. 10/685,705-U.S. publication 20040177387). Aged mice with adeficiency in either of these proteins exhibit a number of features ofARMD including accumulation of lipofuscin in and drusen beneath the RPE,photoreceptor atrophy, and CNV. Methods for testing the efficacy of acandidate agent using this mouse model are disclosed in U.S. publicationno. 20040177387. In general, a candidate agent is administered to themouse either before or after development of features of ARMD, and atleast one eye is monitored for development or regression of drusenand/or lipofuscin accumulation therein, for affect of the candidateagent on Bruch's membrane, affect on retinal degeneration, and/or foraffect on choroidal neovascularization.

The candidate agent can be administered systemically or locally. Theagent can be delivered orally, intravenously, intraperitoneally,intravitreously, transsclerally or topically. The agent can be deliveredby intravitreal injection, transclerally, by sustained release implant,etc. The eye can be analyzed by ophthalmoscopy, angiography,histopathology or a combination thereof. Any of these methods can beused to assess efficacy of a candidate agent in any animal model. Modelsalso exist for diabetic retinopathy. These examples are but a few of themodel systems in which efficacy of the compounds of the invention can beassessed.

Compounds that show promising results in animal studies are tested inhumans, e.g., using standard protocols and endpoints for clinical trialsfor therapies for ARMD or diabetic retinopathy.

In addition to protocols and endpoints that have typically been employedin evaluating therapies for ARMD, the present invention contemplatestesting the inventive compositions to establish their utility ininhibiting progression from the dry form of ARMD to the wet form.Accordingly, in some embodiments the compositions are administered tosubjects who have been diagnosed with the dry type of ARMD. The abilityof the composition to inhibit progression of the dry form of ARMD to wettype ARMD is assessed.

In one example, subjects with the dry type of ARMD are divided into twogroups. One group receives a single retrobulbar or sub-Tenon injectionof the inventive composition in the vicinity one eye, e.g., in closeproximity to the posterior segment of the eye, while the other groupeither receives either no treatment or a single intravitreal injectionof another therapeutic agent such as Macugen or Lucentis into one eye.The groups are monitored over a period of 6 months to 2 years todetermine the percentage of subjects that progress to the wet type ofARMD.

In another example, subjects with the dry type of ARMD are divided intotwo groups. One group receives a single retrobulbar or sub-Tenoninjection of the inventive composition in the vicinity of one eye, e.g.,in close proximity to the posterior segment of the eye, every 6 monthswhile the other group either receives either no treatment or a singleintravitreal injection of another therapeutic agent such as Macugen orLucentis into one eye every 6 months. The groups are monitored over aperiod of 1-2 years (or longer) to determine the percentage of subjectsthat progress to the wet type of ARMD. In another non-limiting example,subjects with dry type ARMD are divided into two groups. One groupreceives a retrobulbar or sub-Tenon injection of the inventivecomposition in the vicinity of one eye, e.g., in close proximity to theposterior segment of the eye, every 3-6 months while the other groupreceives either no treatment or receives treatment with Macugen orLucentis according to the protocols used for treating wet type ARMD,i.e., intravitreal injection every 4-6 weeks. The groups are monitoredfor a period of 1-2 years (or longer) to determine the percentage ofsubjects that progress to the wet form of ARMD.

In another example the ability of an inventive composition to inhibitprogression of early ARMD (AREDS 2) to intermediate ARMD (AREDS 3) isassessed. Subjects with early ARMD are divided into two groups, one ofwhich receives an inventive composition as described in either of thetwo examples above while the other receives either no therapy or analternative therapy such as Lucentis or Macugen as described in eitherof the two examples above. The groups are monitored for a period of time(e.g., as described above) to determine the percentage of subjects thatprogress from early to intermediate ARMD.

In another example the ability of an inventive composition to inhibitprogression of intermediate ARMD (AREDS 3) to advanced ARMD (AREDS 4) isassessed. Subjects with intermediate ARMD are divided into two groups,one of which receives an inventive composition as described in either ofthe two examples above while the other receives either no therapy or analternative therapy such as Lucentis or Macugen as described in eitherof the two examples above. The groups are monitored for a period of time(e.g., as described above) to determine the percentage of subjects thatprogress from intermediate to advanced ARMD.

In addition to monitoring progression of ARMD, the incidence of sideeffects and complications may also be monitored. Consideration of sideeffects is an important aspect when evaluating the overall outcome andrisk/benefit ratio of a therapy. For example, if two therapies areequally efficacious in terms of inhibiting progression of or treatingARMD, the therapy with a lower incidence of side effects is typicallypreferred for most subjects. In certain embodiments of the inventiontherapy of a macular degeneration related condition such as ARMD, or CNVor RNV from any cause, using a composition of the invention isassociated with fewer side effects over time (e.g., over a 1-2 yearperiod) than therapy with FDA-approved therapy for ARMD.

Identifying Subjects and Assessing Response

The methods of the invention may include providing a subject to which acomposition of the invention is to be administered. The subject istypically at risk of or suffering from an eye disorder characterized bymacular degeneration, choroidal neovascularization, retinalneovascularization, or any combination of these. The composition isadministered to the subject with the intent of treating or preventingsuch condition. Thus the subject will typically have been identified asbeing at risk of or suffering from such a condition. Methods fordiagnosis of macular degeneration and choroidal neovascularization andfor assessing response to therapy are known in the art. Any suitabletests and criteria can be used to identify a subject at risk of orsuffering from a macular degeneration related condition, diabeticretinopathy, or choroidal neovascularization and/or to measure aresponse to therapy. Visual acuity can be measured using, for example, aSnellen chart, a Bailey-Lovie chart, a decimal progression chart, aFreiburg visual acuity test, a measurement of minimum angle ofresolution (MAR) etc. Metamorphopsia (visual distortion) may be measuredusing an Amsler chart. Contrast sensitivity may be measured using aPelli-Robson chart. Diagnostic studies include, but are not limited to,standard ophthalmologic examination of the fundus, stereo biomicroscopicexamination of the macula, intravenous fundus fluorescein angiography,fundus photography, indocyanine green video-angiography, and opticalcoherence tomography. A subject displaying an abnormality on one or moreof these diagnostic studies (e.g., a subject that falls outside a rangethat is considered normal for a healthy eye) may be treated inaccordance with the present invention. Subjects may be classified ashaving early, intermediate, or advanced ARMD in accordance with theclassification scheme used in the Age-Related Eye Diseases Study(AREDS), which is set forth in guidelines developed American Academy ofOphthalmology (American Academy of Ophthalmology, Age Related MacularDegeneration Preferred Practice Pattern™, 2003; available for downloadat URL www. followed immediately byaao.org/aao/education/library/ppp/amd_new.cfm). A subject falling intoany of these categories may be treated in accordance with the presentinvention. If the subject has already developed CNV, the subject mayhave classic CNV, occult CNV, or a mixture of the two. Of coursealternate classification schemes, of which a variety is described in theliterature, could also be used.

ARMD is known to have a genetic component, based on studies showing anincreased incidence of ARMD in individuals with relatives suffering fromARMD (e.g., twin studies). Therefore, a subject may be considered atrisk of developing ARMD if he or she has one or more close relatives(e.g., parent, grandparent, sibling, cousin, uncle, aunt), who hasreceived a diagnosis of ARMD. Individuals who smoke and/or consume ahigh fat diet are also at increased risk. The incidence of ARMDincreases with age. Therefore, an individual over approximately 50 yearsof age, generally at least 60 or at least 70 years of age may beconsidered at increased risk. An individual having drusen and one ormore additional risk factors may be at particular risk for developingARMD. An individual with multiple drusen, particularly if large and withindistinct borders, may be at particular risk. An individual with RPEhyperpigmentation or hypopigmentation or geographic atrophy may be atparticular risk. Specific genetic mutations are associated with variousless common macular degeneration related conditions. A subject who hasreceived a diagnosis of diabetes is at risk of developing diabeticretinopathy.

Response to therapy can be assessed by any of the methods mentionedabove. Numerous studies have been conducted to assess the efficacy of avariety of different therapies in restoring vision, preventing visualloss, and/or resulting in improvement or slowing progression of ARMD orchoroidal neovascularization as judged by diagnostic tests such as thosedescribed above. One of ordinary skill in the art will be able to selectappropriate criteria by which to judge the efficacy of therapy.

Therapeutic Applications

The compositions of the invention can be administered to a subject totreat a macular degeneration related condition (e.g., ARMD), diabeticretinopathy, retinopathy of prematurity, persistent hyperplasticvitreous syndrome, choroidal neovascularization, etc. The subject mayhave exudative or nonexudative ARMD. In certain embodiments of theinvention that subject has exudative ARMD but does not have RAP while inother embodiments the subject does have RAP.

One particularly advantageous use for the compositions and methods ofthe invention is to inhibit progression of non-exudative ARMD toexudative ARMD or to inhibit progression of non-exudative ARMD to a moresevere form. For example, in certain embodiments of the invention aninventive composition inhibits progression of early ARMD (AREDS 2) tointermediate ARMD (AREDS 3) or to advanced ARMD (AREDS 4). In certainembodiments of the invention the composition inhibits progression ofintermediate ARMD (AREDS 3) to advanced ARMD (AREDS 4). Any of thecompositions of the invention may be used for these purposes in variousembodiments of the invention. In a specific embodiment a composition ofthe invention, e.g., a gel-forming composition of the invention, is usedfor treating subjects with non-exudative ARMD, e.g., to prevent orinhibit progression to exudative ARMD. In certain embodiments thesubject has not developed detectable CNV and the composition prevents ordelays the development of CNV. For example, the subject may have dryARMD, and the composition prevents or delays the onset of wet ARMD. Incertain embodiments the subject has developed detectable CNV and thecomposition slows the rate of progression of CNV and/or causesregression of existing CNV. In certain embodiments the subject has notdeveloped detectable RNV and the composition prevents or delays thedevelopment of RNV. In certain embodiments the subject has developeddetectable RNV and the composition slows the rate of progression of RNVand/or causes regression of existing RNV. The composition can beadministered once or multiple times to a subject who does or does nothave a condition such as CNV or RNV (or both), e.g., at approximatelypredetermined time intervals such as, for example, approximately every 4weeks, approximately every 6 weeks, approximately every 8, 10, 12, 16,20, 24 weeks, approximately every 6, 8, 10, or 12 months, etc. It willbe understood that in any of the methods of this invention, thecomposition should be administered in an amount effective to achieve theindicated result, within sound medical judgment. It should also beunderstood that the result need not be achieved in every instance.

Ancillary therapies may also be used concurrently, prior to, orfollowing treatment using the compositions and methods of the invention.Such therapies include, but are not limited to, administration ofantioxidant vitamin and/or mineral therapy, photodynamic therapy (e.g.,with verteporfin or other agents), administration of antiinflammatoryagents, antiangiogenic therapy (e.g., administration of one or moreangiogenesis inhibitors such as anecortave acetate or other angiostaticsteroids; anti-VEGF or anti-VEGFR antibody, antibody fragment, siRNA,antisense RNA, or aptamer; or any other antiangiogenic agent includingbut not limited to a small molecule, siRNA, antisense RNA, or aptamertargeted to any proangiogenic gene), growth factor administration,implantation of cells (e.g., neural stem cells, RPE stems cells, RPEcells) into the eye, laser photocoagulation, radiation therapy, thermaltherapy, and surgery (e.g., submacular surgery or maculartranslocation). In certain embodiments of the invention a growth factorfor RPE cells is administered, e.g., REF-1/TFPI-2 (Tanaka, Y, et al.,Invest Ophthalmol Vis Sci. 45(1):245-52, 2004).

It may be desirable to treat an eye that already suffers from choroidaland/or retinal neovascularization (e.g., in a subject with diabeticretinopathy or ARMD) using photocoagulation or surgery and to alsoadminister a composition of the invention to the subject to preservevision in the other eye and/or prevent a recurrence of CNV and/or RNV inthe eye treated with photocoagulation or surgery.

EXAMPLE Example 1 Prevention of Choroidal Neovascularization in a MouseModel by Administration of VCP

Recombinant VCP is produced in and purified from a Pichia pastorisexpression system as described in Sahu, 1998. VCP is dissolved inphysiological saline at various concentrations.

Mice (3 groups, N=10 in each group) are anesthetized and their pupilsdilated. Krypton red laser photocoagulation is used to generate multiple(e.g., 3-20) laser spots in each eye as described in Bora, 2003. For thefirst group, various doses of VCP (e.g., 0.01-100 μg) are administeredby injection to one eye in each mouse at days 1 and 4 following lasertreatment. The other eye serves as a control. For the second group,various doses of VCP (e.g., 0.01-100 mg/kg) are administeredintravenously. Mice in the third group serve as a control. In anotherexperiment, various doses of VCP are administered at different timepoints following laser treatment. 3

Mice are sacrificed at various time points, perfused with salinecontaining fluorescein-labeled dextran (FITC-dextran, Sigma), their eyesenucleated, and scleral-choroidal-RPE flat mounts are prepared andstained with a mAB against elastin (Sigma) and a CY3-conjugatedsecondary antibody (Sigma) and examined with a confocal microscope andwith light microscopy. The CNV stains green, while elastin in Bruch'smembrane stains red. The number of CNV-positive laser spots isdetermined. A reduced number of spots in the treated eyes or mice versusuntreated eyes or mice indicates that the recombinant VCP is effectivein preventing and/or treating CNV. Flat mounts are also stained forvarious drusen constituents and lipofuscin. Expression of various drusenconstituents and growth factors thought to play a role in CNV isassessed in samples obtained from the eyes by RT-PCR and ELISA assay.

Example 2 Prevention of Choroidal Neovascularization in a Mouse Model byAdministration of VCP

Materials and Methods

Recombinant VCP was produced in and purified from a Pichia pastorisexpression system as described in Sahu, 1998. VCP was dissolved inphysiological saline at various concentrations.

CNV Induction in Mice

C57BL/6 mice (The Jackson Laboratory) were anesthetized with a mixtureof ketamine/xylazine (8:1) and the pupils were dilated with a singledrop of 1% tropicamide. Krypton red laser photocoagulation (50-μm spotsize, 0.05s duration, 250 mW) will be used to generate laser spots inbothounding the optic nerve by using a hand-held coverslip as a contactlens. Formation of a bubble at the laser spot indicated rupture ofBruch's membrane. Multiple laser spots were generated in each eye.

Injection of VCP in the Eyes of Mice

Mice in which CNV has been previously laser-induced were administeredVCP solutions by intravitreal injection. Different groups of mice wereinjected with different quantities of this molecule or of mouse albumin(as a control) to determine the effect of dosage on the efficacy andtoxicity of VCP. Briefly, after anesthesia and dilation of the pupil,the anterior chamber was entered via the limbus with a 28-gauge needleto decompress the eye. Under an operating microscope, which allowsvisualization of the retina, a 32-gauge (blunt) needle was passedthrough a scleral incision, just behind the limbus, into the vitreouscavity. A Hamilton syringe was used to inject 1 μl of a solutioncontaining either VCP or albumin.

Determination of Incidence and Size CNV

Seven days after CNV induction incidence of CNV was determined. Briefly,the mice were perfused with a FITC-dextran (Sigma-Aldrich) solution justprior to sacrifice. After the eyes were excised and fixed for 1 h in 10%phosphate-buffered formalin, RPE-choroid-scleral flat mounts wereprepared as follows. The cornea and the lens were removed and theneurosensory retina carefully dissected from the eyecup. Five radialcuts were from the edge of the eyecup to the equator; thesclera-choroidretinal pigment epithelium (RPE) complex was flat-mounted,with the sclera facing down, on a glass slide in Aquamount. The flatmounts were stained with an anti-elastin specific monoclonal antibody(Sigma-Aldrich) and then with a CY3-conjugated secondary antibody(Sigma-Aldrich) at a suitable concentration, e.g., at a 1/200 dilutionof a 1.0 mg/ml stock solution. Mounts were observed under confocalmicroscopy (LSM510, Zeiss). The prominent neovascular growth stainedgreen whereas the underlying elastin in the Bruch's membrane stained redwithin a laser spot. Images were analyzed with the image analysissoftware AxioVision (Zeiss). The amount of CNV was determined bymeasuring the total green-fluorescent surface area in each picture. Amean green-fluorescent area was obtained for the various groups andcompared using student t-test for comparisons between groups and ANOVAfor comparison among multiple groups. The number of spots studied was asfollows: No treatment control: 35 spots); mouse albumin control: 12spots; VCP (10 μg): 26 spots; VCP (30 μg): 14 spots. Deposition of avariety of different complement components is also measured usingimmunological techniques and/or RT-PCR.

Results

The effects of VCP on the development of CNV was tested in a murinemodel of laser-induced CNV. Briefly, VCP (either 10 μg/eye or 30 μg/eye)was injected in the vitreous 24 hrs after laser induction. Seven daysafter CNV induction, incidence of CNV was determined. Just prior tosacrifice, the mice were perfused with a FITC-dextran (Sigma-Aldrich)solution. After the eyes were excised and fixed in 10%phosphate-buffered formalin, RPE-choroid-scleral flat mounts wereprepared and stained with an anti-elastin specific monoclonal antibody(Sigma-Aldrich) and then with a CY3-conjugated secondary antibody(Sigma-Aldrich). Mounts were observed under confocal microscopy (LSM510,Zeiss). The prominent neovascular growth stained green whereas theunderlying elastin in the Bruch's membrane stained red within a laserspot (FIG. 7). Images were analyzed with the image analysis softwareAxioVision (Zeiss). The amount of CNV was determined by measuring thetotal green-fluorescent surface area in each picture. A meangreen-fluorescent area was obtained for both groups and compared usingstudent t-test for comparisons between groups and ANOVA for comparisonamong multiple groups. Results are described in Table 2 and in the graphin FIG. 8. As is evident both from the table and the graph,administration of 30 μg VCP caused a statistically significant reductionin the mean area of CNV relative to either no treatment oradministration of albumin.

TABLE 2 Effect of VCP on Development of CNV in a Mouse Model MultipleComparisons Dependent Variable: FldAreaGreen Tamhane Mean 95% ConfidenceInterval (I) Group (J) Group Difference (I − J) Std. Error Sig. LowerBound Upper Bound No-treatment control No-treatment control MouseAlbumin Control 1909.00993 1090.44317 .601 −1325.1110 5143.1309 VCP 10ug −666.84488 1151.10903 1.000 −4015.6447 2681.9549 VCP 30 ug4877.53314* 848.72770 .000 2345.4558 7409.6105 Compstatin 30 ug5194.92113* 846.05120 .000 2668.9363 7720.9060 Mouse Albumin ControlNo-treatment control −1909.00993 1090.44317 .601 −5143.1309 1325.1110Mouse Albumin Control VCP 10 ug −2575.85481 1053.13838 .182 −5733.0303581.3207 VCP 30 ug 2968.52321* 710.20220 .013 533.9807 5403.0657Compstatin 30 ug 3285.91120* 707.00147 .006 853.1175 5718.7049 VCP 10 ugNo-treatment control 666.84488 1151.10903 1.000 −2681.9549 4015.6447Mouse Albumin Control 2575.85481 1053.13838 .182 −581.3207 5733.0303 VCP10 ug VCP 30 ug 5544.37802* 800.23300 .000 3101.1268 7987.6293Compstatin 30 ug 5861.76601* 797.39374 .000 3424.3034 8299.2287 VCP 30ug No-treatment control −4877.53314* 848.72770 .000 −7409.6105−2345.4558 Mouse Albumin Control −2968.52321* 710.20220 .013 −5403.0657−533.9807 VCP 10 ug −5544.37802* 800.23300 .000 −7987.6293 −3101.1268VCP 30 ug Compstatin 30 ug 317.38799 176.41849 .574 −214.1787 848.9547Compstatin 30 ug No-treatment control −5194.92113* 846.05120 .000−7720.9060 −2668.9363 Mouse Albumin Control −3285.91120* 707.00147 .006−5718.7049 −853.1175 VCP 10 ug −5861.76601* 797.39374 .000 −8299.2287−3424.3034 VCP 30 ug −317.38799 176.41849 .574 −848.9547 214.1787Compstatin 30 ug *The mean difference is significant at the .05 level.

Example 3 Treatment with SPICE in a Mouse Model of Age Related MacularDegeneration

Expression vectors suitable for expression of recombinant SPICE inmammalian or insect cells are generated, and SPICE is produced in andpurified from 293T cells or Spodoptera frugiperda cells as described inRosengard, 2002.

SPICE is administered to 15, 16, or 18 month old normal mice andage-matched mice deficient in Ccl-2 and/or Ccr-2 (Ambati, et al.).Administration is performed by injection to one eye. The other eyeserves as a control. In another experiment SPICE is administeredintravenously. Various doses (as in Example 1) and treatment regimensare used. For example, in some groups SPICE is injected every 3 days. Inother groups SPICE is injected weekly. The mice are sacrificed atvarious time points and their eyes are processed and analyzed asdescribed in Example 2. The ability of SPICE to prevent, inhibit, and/ortreat CNV or other features of ARMD such as photoreceptor atrophy,drusen and lipofuscin accumulation, etc., is assessed.

Example 4 Prevention of Choroidal Neovascularization in a Mouse Model byAdministration of SPICE

Example 2 is repeated using SPICE instead of VCP. Amounts ranging from1-30 μg are tested.

Example 5 Preparation of Collagen Solutions for a Gel-FormingComposition

Stock Collagen Preparation.

Collagen for all formulations will be prepared from porcine corium.Split porcine hide will be procured from Lampire Biological Laboratories(Pipersville, Pa.). Split hide will be rinsed with reagent alcohol andplaced in frozen storage prior to receipt. Sections of split corium willbe cut into small pieces (about 1 cm²) and soaked in reagent alcohol andthen washed extensively with sterile water. The washed pieces will beplaced in 20 volumes of 0.5M HCl for 30 minutes, washed with sterilewater and then placed in 20 volumes of 0.5N NaOH for 30 minutes. Bothtreatments have been shown to be effective in reducing viral titers byup to 6 logs. In addition, both treatments have been shown to havesignificant bactericidal effects, reducing bacterial loads by up to 9logs. The chemically disinfected corium will be washed extensively insterile water, weighed and placed in 20 volumes (v/w) of 0.5 M aceticacid. The pieces will be stirred for 72 hours and porcine mucosal pepsinadded to the partially swollen corium.

Pepsin will be added at 2% (w/w wet corium) and stirred for 48 hours. Anadditional aliquot of pepsin will be added at 1% (w/w wet corium) andstirred for another 24 hours. At this point, the corium should be“dissolved” in acetic acid. Small, undissolved pieces will be removed byfiltering the thick slurry through cheesecloth. The filtrate will bediluted with 0.5M acetic acid and dialyzed against 0.5N acetic acidusing dialysis tubing having a 50,000 dalton nominal cut-off. Analternate dialysis method will utilize ultrafiltration/diafiltrationcartridges procured from Amersham Biotech. The dialysis process removespepsin and degraded pepsin. The retained liquid containing collagen willbe subjected to differential NaCl precipitation to isolate predominantlyType I collagen. Purified Type I collagen at about 5 mg/mL will be thendialyzed against 0.1N acetic acid to remove residual salts (about 5,000nominal molecular weight cut-off). The retained collagen solution willsubsequently be filtered through 0.45 μm and 0.2 μm filters and placedin sterile, 2-liter glass bottles. Collagen concentration will beapproximately 5 mg/mL. All steps will be conducted at room temperature.Stock solutions will be stored at 2-8° C.

Process Controls and Quality Control Tests: Final Stock Collagen will beExamined by the Following Methods.

Analysis by SDS-PAGE to determine collagen purity;

Analysis of uronic acid to determine amounts of residualglycosaminoglycan

Assay of hydroxyproline to determine total collagen concentration;

Differential Scanning Calorimetry to measure temperature of phasetransition (pure, undenatured telopeptide-poor collagen has a transitiononset of about 39° C.)

Sterility using USP methods

Endotoxin using LAL methods

Preparation of In Situ Gelling Collagen Solutions.

Purified, pepsin-digested collagen will be precipitated by addition ofsolid NaCl to 0.8 M. The resultant precipitate will be recovered bycentrifugation at 3500 RPM, a wet weight determined, and the precipitateplaced in dialysis tubing having a NMW cut-off of 50,000 daltons.Attempts will be made to add enough precipitate to produce finalcollagen solutions at 30 and 50 mg/mL (3 and 5%). The tubing will beplaced in 20 volumes of 0.035 M EDTA in deionized water, pH 5.0 anddialyzed with agitation for 24 hours. At this point, the dialysis tubingwill be transferred to another 20 volumes of 0.035 M EDTA, pH 5.5.Dialysis will be conducted again for 24 hours after which the tubingwill be placed in 0.035 M EDTA, pH 6.0. This sequence will be continueduntil dialysis in a final EDTA solution at pH 7.5. This slow increase inpH during EDTA dialysis results in a collagen preparation that remains“soluble” at neutral pH. This is in contrast to standard collagensolutions that spontaneously undergo fibrillogenesis at neutral pH androom temperature. The neutral pH, EDTA-treated collagen solution willremain in solution during storage and will rapidly undergo gelation andfibril formation when exposed to physiological fluids.

Example 6 In Vitro Release of SPICE from Different Collagen Compositions

The release rates of SPICE from collagen plugs produced from collagenprepared as described in Example 5 will be measured daily for a periodof 150 days. Five different in-situ gel-forming collagen-matrixpreparations will be tested:

Collagen 1: 30 mg/ml (3% w/v) of collagen Type 1

Collagen 2: 50 mg/ml (5% w/v) of collagen Type 1

Collagen 3: 30 mg/ml (3% w/v) of collagen Type 1 mixed with 3 mg/ml offibrillar collagen

Collagen 4: 30 mg/ml (3% w/v) of collagen Type 1 mixed with 6 mg/ml offibrillar collagen

Collagen 5: 50 mg/ml (5% w/v) of collagen Type 1 mixed with 5 mg/ml offibrillar collagen

Each collagen preparation will be mixed with either unmodified SPICE orbiotinylated SPICE at a final concentration ranging from 1-10 mg/ml.Plugs will be generated by injecting 0.5 ml of the collagen/protein (3mg of protein/plug) preparation into the wells of a 24-well platecontaining 1 ml of incubation medium. The incubation medium will beeither PBS or PBS/50% plasma. The plugs will be incubated at 37° C. withconstant agitation. The medium will be replaced every day and the amountof SPICE in the supernatant will be measured.

Levels of SPICE will be measured using standard indirect ELISA forunmodified SPICE and with the EZ Biotin Quantitation Kit (PierceBiotechnology) for biotinylated SPICE. Antibodies to SPICE suitable foruse in the ELISA assay are described in U.S. Pat. No. 6,783,759.

Example 7 Inhibition of CNV by Transscleral Delivery of SPICE

We will test whether SPICE can prevent or inhibit CNV when deliveredtranssclerally using the collagen formulation. Laser induction of CNV inthe Yucatan pig is a well-established model to study exudative ARMD. Alaser is used to create ruptures in Bruch's membrane, which triggerneovascularization in the retina surrounding the ruptured membrane overthe course of 7 days (>95% reliability). Approximately 50 spots per eyecan thus be created and used for analysis. Two eyes in two differentpigs are generally accepted as statistically relevant per study group.Three collagen preparations will be chosen for this experiment.Preferred collagen preparations can be selected by measuring the rate ofrelease of SPICE from collagen plugs when such plugs are incubated invitro in a fluid approximating physiological conditions, e.g., phosphatebuffered saline, optionally including 50% plasma by volume, as describedin the previous example. Preparations that provide sustained amounts ofSPICE for a reasonable period of time (e.g., at least several days) inreasonable concentrations (e.g., at least in the μg/ml range) areselected. Of course all of the formulations can be tested and an optimumformulation selected based upon the results in vivo. Twenty four (48eyes) animals will be allotted in 4 groups (12 eyes/group) as follow:

Group 1: Treatment on day 0, Laser induction of CNV on day 14, CNVincidence measurement on day 21

Group 2: Treatment on day 0, Laser induction of CNV on day 30, CNVincidence measurement on day 37

Group 3: Treatment on day 0, Laser induction of CNV on day 60, CNVincidence measurement on day 67

Group 4: Treatment on day 0, Laser induction of CNV on day 90, CNVincidence measurement on day 97

Of the 12 eyes assigned to each group, two eyes will be randomlyassigned one of the following treatments:

Treatment 1: Collagen preparation 1. Chosen as one of three optimalformulations as determined by earlier experiments.

Treatment 2: Collagen preparation 2. Chosen as one of three optimalformulations as determined by earlier experiments.

Treatment 3: Collagen preparation 3. Chosen as one of three optimalformulations as determined by earlier experiments.

Treatment 4: Retrobulbar SPICE. In this group, pigs will receivebilateral retrobulbar injections of 3 ml of PBS containing 6 mg ofSPICE.

Treatment 5: Intravitreal SPICE. In this group, pigs will receivebilateral intravitreal injections of 500 μl of PBS containing 6 mg ofSPICE

Treatment 6: Positive control. In this group, pigs will be leftpharmacologically untreated in one eye for the duration of the study andsimply receive bilateral retrobulbar injections of 3 ml of PBS. It is apositive control because neovascularization is expected in >95% of casesfollowing laser treatment.

CNV Induction in Pigs.

Ten to twelve weeks old Yucatan pigs (The Jackson Laboratory) will beanesthetized with sodium pentobarbital and the pupils will be dilatedwith a single drop of 1% tropicamide. Krypton red laser photocoagulation(50 μm spot size, 0.05 s duration, 250 mW) will be used to generatelaser spots surrounding the optic nerve by using a handheld coverslip asa contact lens. A bubble should form at the laser spot indicatingrupture of Bruch's membrane. Fifty laser spots will be generated in eacheye. Therefore, 100 laser spots per treatment condition will be studied(50 spots/eye×2 eyes/condition).

Retrobulbar Injection of Collagen/SPICE.

The animals will be anesthetized with sodium pentobarbital. Using a30-gauge needle a subconjunctival injection will be made in the superiortemporal region of the eye, carefully avoiding extraocular muscles.Three ml of either the collagen/SPICE or PBS/SPICE solution will beinjected. Formation of the collagen plug in situ will be evaluated byvisual and tactile inspection.

Intravitreal Injection of SPICE in the Eyes of Pigs.

After anesthesia and dilation of the pupil, the anterior chamber will beentered via the limbus with a 28-gauge needle to decompress the eye.Under an operating microscope, which allows visualization of the retina,a 32-gauge (blunt) needle will be passed through a scleral incision,just behind the limbus, into the vitreous cavity. A Hamilton syringewill be used to inject 500 μl of a SPICE solution.

RPE-Choroid-Scleral Flat Mounts Preparation.

Pigs will be anesthetized (Sodium pentobarbital) and perfused throughwith PBS containing fluorescein-labeled dextran (FITC-Dextran, 2 millionaverage molecular weight, Sigma) prior to sacrifice. The eyes will beremoved and fixed for 1 h in 10% phosphate-buffered formalin. The corneaand the lens will be removed and the neurosensory retina will becarefully dissected from the eyecup. Five radial cuts will made from theedge of the eyecup to the equator; the sclera-choroidretinal pigmentepithelium (RPE) complex will be flat-mounted, with the sclera facingdown, on a glass slide in Aquamount. Flat mounts will be stained with amAb against elastin (Sigma) and a CY3-conjugated secondary antibody(Sigma) and examined with a confocal microscope (Zeiss LSM510). The CNVwill be stained green whereas the elastin in the Bruch's membrane willbe stained red. A laser spot with green vessels will be scoredCNV-positive, and a laser spot lacking green vessels will be scoredCNV-negative.

Determination of Incidence and Size CNV

Seven days after CNV induction, incidence of CNV will be determined.Briefly, the pigs will be perfused with a FITC-dextran (Sigma-Aldrich)solution just prior to sacrifice. After the eyes are excised and fixedin 10% phosphate-buffered formalin, RPE-choroid-scleral flat mounts willbe prepared as described above. The flat mounts will be stained with ananti-elastin specific monoclonal antibody (Sigma-Aldrich) and then witha CY3-conjugated secondary antibody (Sigma-Aldrich). Both antibodieswill be used at a 1/200 dilution of a 1.0 mg/ml stock solution. Mountswill be observed under confocal microscopy (LSM510, Zeiss). Theprominent neovascular growth will stain green whereas the underlyingelastin in the Bruch's membrane will stain red within a laser spot.

Interpretation of Results

For determining incidence of CNV, flat mounts will be observed underconfocal microscopy (LSM510, Zeiss). Size of the CNV complex (e.g., meanCNV area) will be graded by morphometric image analysis (Axiovision;Zeiss). Differences between groups will optionally be evaluated by ANOVAand/or other suitable statistical methods. A statistically significantdifference in the size and/or incidence of CNV complexes, or a trendtowards such a difference, in which the treated eyes show a reduction insize and/or incidence relative to untreated eyes, is an indication ofeffectiveness over the time period tested. For example, a statisticallysignificant difference in the size and/or incidence of CNV complexes, ora trend towards such a difference, in which the treated eyes show areduction in size and/or incidence relative to untreated eyes, when anyof the compositions (treatment 1-5) is administered to Group 4, is anindication that the effect of the composition persists over at least 90days.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims. In the claims articlessuch as “a”, “an” and “the” may mean one or more than one unlessindicated to the contrary or otherwise evident from the context. Claimsor descriptions that include “or” between one or more members of a groupare considered satisfied if one, more than one, or all of the groupmembers are present in, employed in, or otherwise relevant to a givenproduct or process unless indicated to the contrary or otherwise evidentfrom the context. The invention includes embodiments in which exactlyone member of the group is present in, employed in, or otherwiserelevant to a given product or process. The invention also includesembodiments in which more than one, or all of the group members arepresent in, employed in, or otherwise relevant to a given product orprocess. Furthermore, it is to be understood that the inventionencompasses all variations, combinations, and permutations in which oneor more limitations, elements, clauses, descriptive terms, etc., fromone or more of the listed claims is introduced into another claim. Inparticular, any claim that is dependent on another claim can be modifiedto include one or more limitations found in any other claim that isdependent on the same base claim. Furthermore, where the claims recite acomposition, it is to be understood that methods of administering thecomposition according to any of the methods disclosed herein, andmethods of using the composition for any of the purposes disclosedherein are included, and methods of making the composition according toany of the methods of making disclosed herein are included, unlessotherwise indicated or unless it would be evident to one of ordinaryskill in the art that a contradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldit be understood that, in general, where the invention, or aspects ofthe invention, is/are referred to as comprising particular elements,features, etc., certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein.

The inclusion of a “providing” step in certain methods of the inventionis intended to indicate that the composition is administered to treat aneye disorder. Thus the subject will have or be at risk of an eyedisorder and the composition is administered to treat the disorder,typically upon the sound recommendation of a medical or surgicalpractitioner, e.g., an ophthalmologist, who may or may not be the sameindividual who administers the composition. The invention includesembodiments in which a step of providing is not explicitly included andembodiments in which a step of providing is included. The invention alsoincludes embodiments in which a step of identifying the subject as beingat risk of or suffering from a eye disorder characterized by maculardegeneration, CNV, or both, is included.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anyVCCP or VCIP or fragment or variant of either), any method ofadministration, any eye disorder or condition or characteristic(s)thereof, or any subject characteristic(s) can be excluded from any oneor more claims, for any reason, whether or not related to the existenceof prior art.

1. A method of treating an eye disorder characterized by maculardegeneration, choroidal neovascularization, retinal neovascularization,or any combination of these, comprising steps of: (i) providing asubject, wherein the subject is suffering from the eye disorder; and(ii) administering a composition comprising a viral complement controlprotein (VCCP) or VCCP variant comprising a polypeptide at least 90%identical to a VCCP, set forth in SEQ ID NO: 2 to the subject in orderto treat the eye disorder, wherein the composition is locallyadministered to the eye or in the vicinity of the eye by injection,implantation, catheter, or cannula.
 2. The method of claim 1, wherein aVCCP or a VCCP variant comprising polypeptide at least 90% identical toa VCCP is administered.
 3. The method of claim 2, wherein the VCCP is apoxvirus complement control protein (PVCCP).
 4. The method of claim 3,wherein the PVCCP is smallpox inhibitor of complement enzymes (SPICE).5. The method of claim 3, wherein the PVCCP is vaccinia complementcontrol protein (VCP).
 6. The method of claim 1, wherein the compositionis locally administered to the eye.
 7. The method of claim 1, whereinthe composition is locally administered as a liquid.
 8. The method ofclaim 1, wherein the composition is locally administered as an ointmentor gel.
 9. The method of claim 8, wherein the gel is formed prior toadministration and is administered behind the sclera.
 10. The method ofclaim 8, wherein the gel is formed from a composition containing solublecollagen.
 11. The method of claim 1, wherein the composition is locallyadministered in an ocular or periocular implant or insert.
 12. Themethod of claim 1, wherein the composition is locally administered inclose proximity to the posterior segment of the eye.
 13. The method ofclaim 1, wherein the composition is locally administered as a solutionthat forms a gel after introduction into the body.
 14. The method ofclaim 13, wherein the solution comprises soluble collagen.
 15. Themethod of claim 14, wherein the solution further comprises fibrillarcollagen solids.
 16. The method of claim 13, wherein the solution isadministered behind the sclera.
 17. The method of claim 1, wherein thesubject is suffering from age-related macular degeneration (ARMD). 18.The method of claim 1, wherein the subject is suffering from choroidalneovascularization (CNV).
 19. The method of claim 1, wherein the subjectis suffering from retinal neovascularization.
 20. The method of claim 1,wherein the subject is suffering from diabetes.
 21. The method of claim1, wherein the subject has been identified as suffering from the eyedisorder based at least in part on an examination of the fundus of thesubject's eye.
 22. The method of claim 1, wherein the subject has beenidentified as suffering from the eye disorder based at least in part ona test that assesses the subject's eye.
 23. The method of claim 1,wherein the subject has been identified as suffering from the eyedisorder based at least in part on an optical coherence tomographystudy.
 24. A method of treating an eye disorder characterized by maculardegeneration, choroidal neovascularization, retinal neovascularization,or any combination of these, comprising steps of: (i) providing asubject, wherein the subject is suffering from the eye disorder; and(ii) administering a composition comprising a VCCP, VCCP or variantcomprising a sequence at least 90% identical to a VCCP set forth in SEQID NO: 2, to the subject in order to treat the eye disorder, wherein thecomposition further comprises a moiety that binds to a component presentin the eye of a subject suffering from the eye disorder.
 25. The methodof claim 24, wherein the moiety binds to a cellular marker present on orat the surface of an endothelial cell or retinal pigment epithelialcell.
 26. The method of claim 24, wherein the moiety binds to a drusenconstituent.
 27. The method of claim 24, wherein the moiety is linked tothe VCCP or VCIP.
 28. A method of inhibiting neovascularization in theeye of a subject suffering from an eye disorder characterized by maculardegeneration, choroidal neovascularization, retinal neovascularization,or any combination of these, comprising the step of: administering acomposition comprising a VCCP of SEQ ID NO: 2 or a VCCP variantcomprising polypeptide at least 90% identical to a VCCP to or in closeproximity to the posterior segment of the subject's eye by introducingthe composition into or in close proximity to the posterior segment ofthe subject's eye by injection, implantation, catheter, or cannula. 29.The method of claim 28, wherein a VCCP is administered.
 30. The methodof claim 29, wherein the VCCP is SPICE.
 31. The method of claim 29,wherein the VCCP is VCP.
 32. The method of claim 29, wherein the VCCP isa PVCCP.
 33. The method of claim 28, wherein the composition isadministered as a liquid.
 34. The method of claim 28, wherein thecomposition is administered as a gel.
 35. The method of claim 34,wherein the gel is formed prior to administration and is administeredbehind the sclera.
 36. The method of claim 34, wherein the gel is formedfrom a composition containing soluble collagen.
 37. The method of claim28, wherein the composition is administered in an ocular or periocularimplant or insert.
 38. The method of claim 28, wherein the compositionis administered as a solution that forms a gel after introduction intothe body.
 39. The method of claim 38, wherein the solution comprisessoluble collagen.
 40. The method of claim 39, wherein the solutionfurther comprises fibrillar collagen solids.
 41. The method of claim 38,wherein the solution is administered behind the sclera.
 42. The methodof claim 28, wherein the subject is suffering from ARMD.
 43. The methodof claim 28, wherein the subject is suffering from CNV.
 44. The methodof claim 28, wherein the subject is suffering from RNV.
 45. The methodof claim 28, wherein the subject is suffering from diabetes.
 46. Themethod of claim 28, wherein the subject has been identified as sufferingfrom the eye disorder based at least in part on a test that assesses thesubject's eye.
 47. The method of claim 28, wherein the subject has beenidentified as suffering from the eye disorder based at least in part onan examination of the fundus of the subject's eye.
 48. The method ofclaim 28, wherein the subject has been identified as suffering from theeye disorder based at least in part on an optical coherence tomographystudy.
 49. A method of treating an eye disorder characterized by maculardegeneration, choroidal neovascularization, retinal neovascularization,or any combination of these, comprising steps of: (i) providing asubject who is suffering from the eye disorder; and (ii) administering acomposition comprising a VCCP or VCCP variant comprising a sequence atleast 90% identical to a VCCP set forth in SEQ ID NO:
 2. 50. The methodof claim 49, wherein a VCCP is administered.
 51. The method of claim 49,wherein the VCCP is a PVCCP.
 52. The method of claim 51, wherein thePVCCP is SPICE.
 53. The method of claim 51, wherein the PVCCP is VCP.54. The method of claim 49, wherein the subject is suffering from ARMD.55. A method of inhibiting neovascularization in the eye of a subjectsuffering from an eye disorder characterized by macular degeneration,choroidal neovascularization, retinal neovascularization, or anycombination of these, comprising the step of: administering acomposition comprising a VCCP or VCCP variant comprising a sequence atleast 90% identical to a VCCP set forth in SEQ ID NO: 2 to or in closeproximity to the posterior segment of the subject's eye.
 56. The methodof claim 55, wherein a VCCP is administered.
 57. The method of claim 56,wherein the VCCP is SPICE.
 58. The method of claim 56, wherein the VCCPis VCP.
 59. The method of claim 55, wherein the subject is sufferingfrom ARMD.